Serotonin transporter gene and treatment of opioid-related disorders

ABSTRACT

The gene responsible for encoding SERT has a functional polymorphism at the 5′-regulatory promoter region, which results in two forms, long (L) and short (S). The LL-genotype is hypothesized to play a key role in the early onset of alcohol use. The present invention discloses the differences in treatment and diagnosis based on the L or short genotypes as well as on a single nucleotide polymorphism of the SERT gene, the 3′ UTR SNP rs1042173. The present invention demonstrates the efficacy of using the drug ondansetron and similar drugs for treatment based on variations in the polymorphisms of the SERT gene as well as methods for diagnosing susceptibility to abuse of alcohol and other addiction-related diseases and disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application a continuation of U.S. patent application Ser. No.16/133,234, filed on Sep. 17, 2018, which is a continuation of U.S.patent application Ser. No. 15/783,676, filed on Oct. 13, 2017, which isa continuation of U.S. patent application Ser. No. 15/417,933, filed onJan. 27, 2017, which is a continuation of U.S. patent application Ser.No. 15/243,682, filed on Aug. 22, 2016, which is a continuation of U.S.patent application Ser. No. 14/886,691, filed on Oct. 19, 2015, which isa continuation of U.S. patent application Ser. No. 14/189,746, filed onFeb. 25, 2014, which is a divisional of U.S. patent application Ser. No.12/919,905, filed on Jan. 11, 2011, which is a U.S. National StageFiling under 35 U.S.C. 371 from International Application No.PCT/US2009/035420, filed on Feb. 27, 2009, and published as WO2009/108837, which is entitled to priority pursuant to 35 U.S.C. §119(e) to U.S. provisional patent application nos. 61/032,263, filed onFeb. 28, 2008, 61/059,301, filed on Jun. 6, 2008, and 61/146,440, filedon Jan. 22, 2009. The entire disclosures of the afore-mentionedprovisional U.S. patent applications are incorporated herein byreference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos. U10AA011776-10, 1 N01 AA001016-000, 7 R01 AA010522-12, 5 R01 AA012964-06, 5K23 AA000329-06, 3 R01 DA012844 and 5 R01 DA013783 awarded by theNational Institutes of Health. The government has certain rights in theinvention.

FIELD OF INVENTION

This invention relates generally to the field of diagnosing thesusceptibility to addiction-related diseases and disorders and impulsecontrol disorders, particularly alcohol-related diseases and disorders,as well as monitoring and treating the same.

BACKGROUND

Vulnerability to alcohol dependence is heritable, with a rate rangingfrom 0.52 to 0.64 (Kendler, 2001). Despite this high heritability rate,only one marker allele (alcohol-metabolizing aldehyde dehydrogenasegenes) has been identified consistently to be associated with alcoholism(Kranzler et al, 2002). Of the various neurotransmitter systems throughwhich alcohol mediates its effects, the serotonergic system has beenshown to play an important role in alcohol preference and consumption(Johnson, 2004). Synaptic serotonergic neurotransmission is terminatedwhen serotonin (5-HT) is transported back into pre-synaptic neurons by5-HT transporters (5-HTTs) (Talvenheimo and Rudnick, 1980). Therefore, amajor part of the functional capacity of the serotonergic system isregulated by the 5-HTT. Heavy episodic drinking is associated withnumerous psychiatric and general medical conditions causing a majorpublic health burden (Cargiulo, 2007). Several studies have reported adose-response relationship between the extent of heavy drinking and therisk of alcohol related morbidity and mortality among heavy drinkers(Makela and Mustonen, 2007; Gastfriend et al., 2007). Consequently,reduction of heavy drinking is used as an indicator of treatmentresponse in clinical trials aimed at treating alcohol dependence.

Of the various neurotransmitter systems through which alcohol mediatesits effects, the serotonergic system has been shown to play an importantrole in alcohol preference and consumption (Johnson, 2004). Synapticserotonergic neurotransmission is terminated when serotonin (5-HT) istransported back into pre-synaptic neurons by 5-HT transporters (5-HTTs)(Talvenheimo and Rudnick, 1980) and the degree of 5-HT reuptake dependson the density of 5-HTTs on presynaptic surface. The selective 5-HTreuptake inhibitors that act directly on 5-HTTs have been shown toreduce alcohol consumption in rats (Gill and Amit, 1989). However, inhumans SSRIs have been effective at reducing heavy drinking only amongsome subtypes of alcoholics, more specifically in type A alcoholics butnot in type B alcoholics (Dundon et al., 2004; Pettinati et al., 2000)who are considered to be more biologically predisposed to developalcohol dependence. Therefore, it is reasonable to propose that allelicvariations which alter expression levels of SLC6A4 gene can be expectedto have an important effect on drinking intensity.

The human 5-HTT is encoded by a single gene (SLC6A4) mapped onchromosome 17q11.1q12 (Ramamoorthy et at, 1993). The SLC6A4 gene spans˜35 kb and has 14 exons. The protein encoded by this gene, the 5-HTT, isa trans-membrane protein containing 630 amino acids (Heils et al.,1996). The expression level of SLC6A4 is regulated by at least threemechanisms: transcription regulatory elements in the promoter(Ramamoorthy et al., 1993), differential splicing (Bradley and Blakely,1997), and the use of different 3′ polyadenylation sites (Battersby etal., 1999). Furthermore, several other polymorphisms that change aminoacid sequence (Thr4Ala, Gly56Ala, Glu215Lys, Lys605Asn and Pro612Ser) of5-HTT have been shown to affect 5-HT uptake function in cell cultures(Prasad et al., 2003).

Although the long (L) and short (S) polymorphism at 5-HTT linkedpolymorphic region (5-HTTLPR) of SLC6A4 has been extensively studied inthe literature, the results are inconclusive. For example, in ameta-analysis of 17 studies, Feinn et al. (2005) showed that S allelewas significantly associated with alcohol dependence in subjects withco-occurring serotonergic abnormalities while several other studiesreported an association of alcohol dependence with the L allele (Kweonet al., 2005, Hu et al., 2005). On the other hand, numerous studiesincluding the report by our group reveal a differential associationbetween chronic problem-drinking and the density and function ofserotonin transporters in alcoholic subjects carrying L and S variantsof SLC6A4 (Little et al., 1998, Javors et al., 2005, Johnson et al.,2008). Located in the gene's transcriptional control region, 5-HTTLPRcontains 16 tandem repeats of a 20 to 23 bp (G+C)-rich sequence betweenbp −1376 and bp −1027. Two common forms of this transcriptional controlregion have been found: a long 528 bp allele (L) with 16 repeats and ashort 484 bp allele (S) with a deletion of 44 bp extending from bp −1255to bp −1212.

Serotonin (5-HT) function has been implicated in the regulation of mood,impulsivity, and alcohol use that includes variation in the age of onsetof drinking and onset of alcohol use disorders. The 5-HT system,originating in the raphe nuclei and projecting to cortex, hippocampus,and subcortical brain regions, is thought to influence drinking behaviordirectly in alcohol-use-disordered individuals by modulating thereinforcing effects of alcohol and/or indirectly by processes regulatingimpulsivity and affect. Findings from animal studies have shown thatpharmacological enhancement of 5-HT activity inhibits alcohol intake.Human studies have shown that low 5-HT turnover is associated withimpulsivity], as well as alcohol-seeking behavior and alcoholism. Lowercentral 5-HT turnover (e.g., 5-hydroxy indole acetic acid incerebrospinal fluid) has been reported in early-onset alcohol-dependent(EOA) adults compared to late-onset alcohol dependent adults (LOA) andthe lowest central 5-HT turnover occurs in EOA adults when both parentshave alcohol dependence. Together, these findings support the hypothesisthat 5-HT availability and function regulate drinking-related behaviorsand drinking history.

Scientific frustration has been promulgated by failures to demonstrateclinical efficacy for selective serotonin reuptake inhibitors (SSRIs) intreating alcoholism. Animal studies show consistently that SSRIs reducealcohol consumption in various models and across species (for a review,see Johnson and Ait-Daoud 2000). SSRIs augment central serotonergicfunction and, by tonic inhibition, decrease mesocorticolimbic dopamine(DA) release. DA activation mediates alcohol's rewarding effects; hence,its diminution should be associated with decreased abuse liability.Moreover, in humans, there is solid evidence that individuals with thehighest biological predisposition to alcoholism, typically by having anearly disease onset, family history, or both, have reduced serotonergicfunction (Buydens-Branchey et al. 1989; Fils-Aime et al 1996; LeMarquandet al 1994a; LeMarquand et al 1994b; Swann et al 1999). It was,therefore, tempting to predict that alcoholics would benefit from SSRItreatment, and that those with an early onset and/or family historywould benefit the most because the SSRI would presumably ameliorate theexisting disequilibrium in serotonergic function.

Despite the encouraging results of earlier studies, more rigorous, wellcontrolled, state-of-the-art trials have generally failed to find atherapeutic effect for SSRIs in treating alcoholism (Gorelick andParedes 1992; Kranzler et al 1996).

In humans, functional control of the serotonergic system also seems tobe regulated by genetic differences in SERT expression (Meltzer andArora 1988). The SERT possesses the only known functional polymorphismregulating the serotonin system (Heils et al 1997; Heils et al 1996;Lesch et al 1997). Basically, the polymorphism of the SERT 5′ regulatorypromoter region (5′-HTTLPR) on chromosome 17p12 consists of two types(Heils et al 1997; Heils et al 1996; Lesch et al 1997). The long (LL)variant, compared with the short (SS) or heterozygous (SL) form, isassociated with three times greater 5-HT uptake from platelets(Greenberg et al 1999) and in lymphoblasts (Lesch et al 1996). Hence,individuals with the LL variant of 5′-HTTLPR can be expected to haveincreased SERT number and function and reduced levels of intrasynaptic5-HT.

Recent scientific evidence would support the hypothesis of LL variant of5′-HTTLPR predominance among EOA (Ishiguro et al 1999; Schuckit et al1999). Turker et al. (1998) suggested that high ethanol tolerance may beassociated with the SS/SL form of 5′-HTTLPR, but their rather informalcriteria and the use of controls from a blood bank with uncertainalcohol histories may make their conclusions difficult to substantiate.Furthermore, a study by Sander et al. (1998) did not find a significantrelationship (p=0.09) between SS/SL genotype and alcoholics withdissocial personality disorder. Finally, there are conflicting data onthe relationship between the SS/SL form of 5′-HTTLPR and alcoholism ingeneral (Edenberg et al. 1998; Hammoumi et al. 1999; Jorm et al. 1998;Sander et al. 1997); however, these studies contain no subtypinginformation. Moreover, it is difficult to compare these epidemiologicgenotyping studies because of differing diagnostic criteria between thestudies and different population frequencies across ethnic groups forthe allelic forms. Perhaps most importantly, none of these studies havetaken into account that it may be the interaction between these subtypesand alcohol consumption which is critical. That is, even though theseallelic forms of the SERT may not determine vulnerability to alcoholismper se, the interaction between the allelic forms and alcoholconsumption may determine treatment response, particularly to aselective serotonergic agent.

Reduced 5-HT neurotransmission has been reported in those with anincreased propensity for drinking and in alcoholics who exhibitantisocial behaviors (i.e., EOA) (LeMarquand et al 1994a; LeMarquand etal 1994b). These results are consistent with: 1) the demonstration ofincreased 5-HT uptake into presynaptic serotonergic neurons in thebrain, in lymphocytes, and in platelets of alcoholics and theirdescendants (Boismare et al 1987; Ernouf et al 1993; Faraj et al 1997)and 2) SPECT studies in nonhuman primates that had undergone earlyenvironmental stress, showing that increased binding of serotonintransporters is associated with greater aggressiveness and reducedsensitivity to ethanol intoxication (Heinz et al 1998). It would,therefore, be tempting to speculate that this hypo-serotonergic statecould render individuals more vulnerable to experimentation with alcoholearly in life.

Although acute alcohol intake may initially produce some temporaryrelief by increasing brain 5-HT levels, the residual effect is to reduceserotonin function, thereby setting up a vicious cycle (for a review seeLeMarquand et al. (LeMarquand et al 1994a; LeMarquand et al 1994b)).Chronic excessive drinking does not result in sustained increases in5-HT neurotransmission (Branchey et al 1981; Ledig et al 1982; Pohoreckyet al 1978). Reduced SERT density in the raphe nuclei is associated withan early alcoholism onset in violent offenders (Tiihonen et al 1997) andwith the combination of having the LL variant of 5′-HTTLPR and chronicdrinking in both postmortem brains (Little et al 1998) and livingindividuals (Heinz et al 2000). The study of Heinz and colleagues (Heinzet al 2000) showed that individuals with the LL form of 5′-HTTLPR aremore vulnerable to chronic alcohol-induced reductions in SERT density,but their study requires validation in an adequately powered prospectivestudy that contains an equal number of individuals with the LL and SS/SLvariants of 5′-HTTLPR. This would enable confirmation of thedifferential phenotypic expression of these allelic forms. Although itmay, at first, seem paradoxical (i.e., for those with the LL variant of5′-HTTLPR to have both reduced SERT density and decreased serotonergicfunction), it is notable that the SERTs in the raphe are associated withthe regulation of cell firing rates.

There is a long felt need in the art for compositions and methods usefulfor diagnosing, treating, and monitoring alcohol disorders andsusceptibility to alcohol disorders. The present invention satisfiesthese needs.

SUMMARY OF THE INVENTION

The present invention discloses several methods and assays useful fordetermining whether a subject has a predisposition to developing anaddictive disease or disorder, determining whether a subject will beresponsive to particular treatments, and compositions and methods usefulfor treating a subject in need of treatment. For example, the presentinvention encompasses compositions and methods, and combinationsthereof, useful for predicting subjects susceptible to increasedintensity of drinking and useful for predicting useful treatments.

The present invention encompasses compositions and methods useful fortreating subjects who abuse alcohol based on identification of geneticmarkers indicative of a subject being predisposed to severe drinking orbeing more susceptible to alcoholism and problem drinking. The assayscenter on the serotonin system, particularly the serotonin transportergene SLC6A4, its expression, and various polymorphisms of that gene. Inone aspect, the marker is based on measurement of nucleotidepolymorphisms. In one aspect, the polymorphism is a single nucleotidepolymorphism (SNP). The invention further provides for the use ofcombinations of assays to help further predict a predisposition todeveloping an addictive disease or disorder and to help predicttreatments based on the results of the assays. In one aspect, at leastone drug which regulates part of the serotonin system is administered tothe subject. In another aspect, combination therapy can be used byadministering additional drugs.

Subjects comprising the G allele of SNP polymorphism rs1042173 of theserotonin transporter gene SLC6A4 were found herein to be associatedwith significantly lower drinking intensity compared to subjectshomozygous for the T allele. This was true for whites but not Hispanics.Additionally, the present application discloses that cells transfectedwith the G allele of SNP polymorphism rs1042173 of the serotonintransporter gene SLC6A4 had significantly higher levels of both the mRNAand the serotonin transporter protein compared with cells transfectedwith the T allele. Even among alcohol-dependent G allele carriers forrs1042173, there was less intensity of drinking of compared withalcohol-dependent subject who were homozygous for the T allele. Thepresent application further discloses that alcohol-dependent subjectswith the TT genotype respond better to ondansetron treatment thansimilar subjects with the TG/GG genotype. Therefore, the presentinvention provides compositions and methods useful for predicting apredisposition to an addictive disease or disorder and the severity ofthat disorder, as well a compositions and methods useful for predictingsuitable treatments and treatment regimens for those subjects. Thepresent invention provides that for those homozygous for T, treatmentmay be customized to increase expression of the SLC6A4 gene or itsprotein, or their levels or activity, and that treatments furtherinclude compositions and methods useful for decreasing serotonin levelsor activity.

The present application discloses that youths with the LL genotype ofthe functional polymorphism for the 5′-regulatory promoter region of theSERT gene (5-HTTLPR) had higher levels of SERT, as measured by³H-paroxetine binding and had a significantly earlier age of onset ofdrinking. The present invention therefore encompasses a method ofpredicting subjects with a predisposition to early onset of drinking aswell as methods of treating these subjects, including treatments toreduce expression of SERT and it activity.

The present application further discloses that the “interaction” oftreatment (with ondansetron) and genotype (LS vs. LS/SS) is highlysignificant and that there is a significant effect of age of onset ofdrinking. The application discloses a significantly higher paroxetinebinding (density of SERT) in LL-genotype vs. S-carriers (SS or SLgenotypes). The present further application discloses that the LL grouphad a significantly earlier age of onset of drinking and a longerduration of drinking. These promising data provide the first evidencethat alcoholics with the LL genotype, compared with their LS/SScounterparts, experience significantly greater reduction in the severityof drinking following ondansetron treatment.

In one embodiment, the present invention provides for treatingalcoholics, as well as subjects with other addictive diseases anddisorders, with at least one drug. In one aspect, the subject has thegenotype LL. In one aspect, the at least one drug is ondansetron. In oneaspect, the treatment reduces DDD. The present invention furtherencompasses the use of multiple drugs and combinations of drugs fortreating subjects described herein.

Furthermore, the present invention provides for the use of combinationsof assays to better predict or diagnose a susceptibility to developingan addictive disease or disorder as well as methods of predicting apersonalized treatment based on the use of one or more predictiveassays. Based on the results of one or more of the assays in a subject,treatments can be designed specifically for that subject.

The present invention encompasses an approach that combines drugs forthe treatment or prevention of addictive disorders such as alcoholdependence. Because the reinforcing effects of most abused drugs arealso mediated by CMDA neurons, the present invention providescombination therapy with drugs such as topiramate, ondansetron, andnaltrexone as efficacious treatments for addictive disorders including(but not limited to) alcohol, eating, cocaine, methamphetamine,marihuana, tobacco abuse and addiction, and other addictive behaviors,including, but not limited to, gambling and sex. Based on the unexpecteddiscoveries described herein, one of ordinary skill in the art will nowappreciate that the compounds of the invention useful for combinationdrug therapy can in some instances be used singly instead of as part ofa combination. Additionally, based on the present application, one ofordinary skill in the art will also appreciate that the compounds of theinvention useful for combination drug therapy can in some instances beused in any combination.

In one embodiment, the present invention provides compositions andmethods for treating or preventing an alcohol-related disease ordisorder comprising administering to a subject a therapeuticallyeffective amount of at least two anti-alcohol agents or compounds, andoptionally other therapeutic agents. Preferably, at least threeanti-alcohol agents or compounds are used in the combination therapy.The present invention further encompasses the adjunctive use ofpsychosocial management techniques. In one aspect, the drug combinationtherapy is more effective alone than when combined with psychosocialmanagement techniques. In another aspect, the drug combination therapycombined with psychosocial management techniques is more effective thandrug combination therapy alone. In one aspect, the present inventionprovides methods for treating or preventing an alcohol-related diseaseor disorder in a subject comprising administering an effective amount ofat least two compounds, or preferably at least three compounds, oranalogs, homologs, derivatives, modifications, and pharmaceuticallyacceptable salts thereof, selected from the group consisting ofserotonergic agents, serotonin antagonists, selective serotoninre-uptake inhibitors, serotonin receptor antagonists, opioidantagonists, dopaminergic agents, dopamine release inhibitors, dopamineantagonists, norepinephrine antagonists, GABA agonists, GABA inhibitors,GABA receptor antagonists, GABA channel antagonists, glutamate agonists,glutamate antagonists, glutamine agonists, glutamine antagonists,anti-convulsant agents, NMDA-blocking agents, calcium channelantagonists, carbonic anhydrase inhibitors, neurokinins, smallmolecules, peptides, vitamins, co-factors, anti-orexin agents,regulators of cannabinoid receptor-1, and Corticosteroid ReleasingFactor antagonists. In one aspect, the neurokinin is NPY. The presentinvention further encompasses administering other small molecules andpeptides.

In one embodiment, the alcohol-related disease or disorder being treatedincludes, but is not limited to, early-onset alcoholic, late-onsetalcoholic, alcohol-induced psychotic disorder with delusions, alcoholabuse, excessive drinking, heavy drinking, problem drinking, alcoholintoxication, alcohol withdrawal, alcohol intoxication delirium, alcoholwithdrawal delirium, alcohol-induced persisting dementia,alcohol-induced persisting amnestic disorder, alcohol dependence,alcohol-induced psychotic disorder with hallucinations, alcohol-inducedmood disorder, alcohol-induced or associated bipolar disorder,alcohol-induced or associated posttraumatic stress disorder,alcohol-induced anxiety disorder, alcohol-induced sexual dysfunction,alcohol-induced sleep disorder, alcohol-induced or associated gamblingdisorder, alcohol-induced or associated sexual disorder, alcohol-relateddisorder not otherwise specified, alcohol intoxication, and alcoholwithdrawal. In one aspect, the alcohol-related disease or disorder isearly onset alcoholic. In another aspect, the alcohol-related disease ordisorder is late onset alcoholic.

In one embodiment, the present invention provides compositions andmethods for reducing the frequency of alcohol consumption compared withthe frequency of alcohol consumption before the treatment. One ofordinary skill in the art will appreciate that the frequency can becompared with prior consumption by the subject or with consumption by acontrol subject not receiving the treatment. In one aspect, the type ofalcohol consumption is heavy drinking. In another aspect, it isexcessive drinking.

In one embodiment, the present invention provides compositions andmethods for reducing the quantity of alcohol consumed in a subjectcompared with the amount of alcohol consumed before the treatment orcompared with the alcohol consumption by a control subject not receivingthe treatment.

One of ordinary skill in the art will appreciate that in some instancesa subject being treated for and addictive disorder is not necessarilydependent. Such subjects include, for example, subjects who abusealcohol, drink heavily, drink excessively, are problem drinkers, or areheavy drug users. The present invention provides compositions andmethods for treating or preventing these behaviors in non-dependentsubjects.

In one embodiment of the invention, the present invention providescompositions and methods for improving the physical or psychologicalsequelae associated with alcohol consumption compared with a controlsubject not receiving the treatment.

In one embodiment, the present invention provides compositions andmethods for increasing the abstinence rate of a subject compared with acontrol subject not receiving the treatment.

In one embodiment, the present invention provides compositions andmethods for reducing the average level of alcohol consumption in asubject compared with the level of alcohol consumption before thetreatment or compared with the level of alcohol consumption by a controlsubject not receiving the treatment.

In one embodiment, the present invention provides compositions andmethods for reducing alcohol consumption and for increasing abstinencecompared with the alcohol consumption by the subject before treatment orwith a control subject not receiving the treatment.

In one embodiment, the present invention provides compositions andmethods for treating a subject with a predisposition to early-onsetalcoholism.

In one embodiment, the present invention provides compositions andmethods for treating a subject with a predisposition to late-onsetalcoholism.

One of ordinary skill in the art will appreciate that there are multipleparameters or characteristics of alcohol consumption which maycharacterize a subject afflicted with an alcohol-related disease ordisorder. It will also be appreciated that combination therapies may beeffective in treating more than one parameter, and that there aremultiple ways to analyze the effectiveness of treatment. The parametersanalyzed when measuring alcohol consumption or frequency of alcoholconsumption include, but are not limited to, heavy drinking days, numberof heavy drinking days, average drinking days, number of drinks per day,days of abstinence, number of individuals not drinking heavily orabstinent over a given time period, and craving. Both subjective andobjective measures can be used to analyze the effectiveness oftreatment. For example, a subject can self-report according toguidelines and procedures established for such reporting. The procedurescan be performed at various times before, during, and after treatment.Additionally, assays are available for measuring alcohol consumption.These assays include breath alcohol meter readings, measuring serum CDTand GGT levels, and measuring 5-HTOL urine levels.

The present invention further provides adjunctive therapies to be usedin conjunction with the combination drug therapies. The presentinvention further provides adjunctive therapy or treatment wherein thesubject is also submitted to a psychosocial management program.Psychosocial management programs are known in the art and include, butare not limited to, Brief Behavioral Compliance Enhancement Treatment,Cognitive Behavioral Coping Skills Therapy, Motivational EnhancementTherapy, Twelve-Step Facilitation Therapy (Alcoholics Anonymous),Combined Behavioral Intervention, Medical Management, psychoanalysis,psychodynamic treatment, and Biopsychosocial, Report, Empathy, Needs,Advice, Direct Advice and Assessment. The present invention furtherencompasses the use of additional adjunct therapies and treatment,including hypnosis and acupuncture.

The present invention further provides for advice to be provided tosubjects in conjunction with drug combination therapy. Adviceconstitutes a set of instructions pertaining to the potentialconsequences of excessive drinking, a calendar or other method formonitoring drinking, and instructions or suggestions about how to reduceor stop drinking. Any of these strategies either alone or in anycombination, and no matter how brief or lengthy, can constitute advice.The advice can be provided in a format such as written, electronic, orinterpersonal. In one embodiment, the drug combination therapy is moreeffective at treatment or prevention than merely administering a placeboand providing advice, administering no drugs and providing advice, ornot administering drugs or providing advice. In one aspect, thecombination drug therapy is more effective at treatment or preventionthan drug therapy used in combination with a psychosocial managementprogram.

In one embodiment, at least one of the compounds being administered isadministered at least once a day. In one aspect, it is administered atleast twice a day. In another embodiment, it is administered at leastonce a week. In yet another embodiment, it is administered at least oncea month.

In one embodiment, at least one of the compounds is a serotonin receptorantagonist. In one aspect, the serotonin receptor is the serotonin-3receptor. In one aspect, the compound is ondansetron.

Various aspects and embodiments of the invention are described infurther detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Haploview generated LD plots for the five SNPs examined in thisstudy and 5-HTTLPR alleles of SLC6A4 gene. The pooled sample consists ofsubjects of Caucasian and Hispanic origin. Number in each box representsD′ values for each SNP pair.

FIG. 2. Amounts of drinking in 165 Caucasian male and female alcoholics.(A) Amounts of drinking as a function of TT, TG and GG genotypes ofrs1042173 (N of subjects in each group is: 47 TT, 77 TG, and 41 GG).Mean drinks per drinking day (±SEM) for the TT, TG and GG subjects were11.17±0.98 vs. 8.05±0.47 and 9.58 ⊥ 0.67, respectively (F=5.63;p=0.004). (B) Drinks per drinking day variance as a function of the TTand G carriers (N of subjects in each genotype is: 47 TT, 118 Gcarriers). Mean drinks per drinking day (±SEM) for the T homozygotes andG carriers were 11.17±0.98 vs. 8.58±0.39 respectively (t=2.97; p=0.003).

FIG. 3. (A) Serotonin transporter (5-HTT) mRNA expression levels in HeLacell cultures quantified by quantitative real-time PCR assay. The datashown here are mean±SEM of four replicates for 5-HTT mRNA expressed bythe T and G alleles in three separate experiments (Exp.) conducted onseparate times. GAPDH=glyceraldehyde-3-phosphate dehydrogenase. (B)Average differences in optical densities of bands seen on immunoblotsfor serotonin transporter (5-HTT) protein expression in HeLa cellcultures for the T and G allele specific expression in three cellcultures (G: 1.23+0.07; T: 0.28+0.05; N=4).

FIG. 4 is a graphic representation of platelet paroxetine binding (Boraxfor LL and S-carrier genotypes (5-HTTLPR genotype). The ordinateindicates platelet binding (Bmax) and the abscissa indicates thegenotype.

DETAILED DESCRIPTION Abbreviations, Generic Names, and Acronyms

-   5-HT—serotonin-   5-HT₃—a subtype of serotonin receptor, the serotonin-3 receptor-   5-HTOL—5-hydroxytryptophol-   5-HTT—serotonin transporter (also referred to as SERT, 5HTT, HTT,    and OCDI)-   5-HTTLPR—serotonin transporter-linked polymorphic region-   ADE—alcohol deprivation effect-   ADI—adolescence diagnostic interview-   ASPD—antisocial personality disorder-   AUD—alcohol use disorder-   BBCET—Brief Behavioral Compliance Enhancement Treatment-   BED—binge eating disorder-   b.i.d.—twice a day-   B_(max)—maximum specific paroxetine binding density-   BRENDA—Biopsychosocial, Report, Empathy, Needs, Direct advice, and    Assessment-   CBI—combined behavioral intervention-   CBT—Cognitive Behavioral Coping Skills Therapy, also referred to as    cognitive behavioral therapy-   CDT—carbohydrate-deficient transferrin-   ChIPS—children's interview for psychiatric syndrome-   CMDA—corrico-mesolimbic dopamine-   DA—dopamine-   DDD—drinks/drinking day-   DSM—Diagnostic and Statistical Manual of Mental Disorders-   EOA—early-onset alcoholic(s)-   G2651T—a site within a putative polyadenylation signal for a    commonly used 3′ polyadenylation site of the SLC6A4 gene; also has    reference identification number rs1042173 at the GenBank website of    the National Center for Biotechnology Information-   GABA—γ-amino-butyric acid (also referred to as γ-amino butyric acid    and γ-aminobutyric acid)-   GGT—γ-glutamyl transferase-   ICD—impulse control disorder-   IP—intraperitoneal-   K_(d)—affinity constant-   K_(m)—equilibrium constant-   L—long-   LOA—late-onset alcoholic(s)-   MET—Motivational Enhancement Therapy-   miRNA—micro RNA-   MM—Medical Management-   NAc—nucleus accumbens-   Naltrexone—a μ opioid receptor antagonist-   ncRNA—non-coding RNA-   NMDA—N-methyl-D-aspartate-   NOS—not otherwise specified-   Ondansetron (Zofran®)—a serotonin receptor antagonist-   P—alcohol-preferring rats-   S—short-   SERT—serotonin transporter (also referred to as 5-HTT)-   SLC6A4—human 5-HT transporter gene,-   SNP—single nucleotide polymorphism-   SSRI—selective serotonin re-uptake inhibitor-   Topiramate (Topamax®)—an anticonvulsant-   TSF—Twelve-Step Facilitation Therapy (e.g., Alcoholics Anonymous)-   V_(max)—maximum serotonin uptake velocity-   VTA—ventral tegmental area    Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. As used herein, each of the followingterms has the meaning associated with it in this section. Specific andpreferred values listed below for radicals, substituents, and ranges arefor illustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents.

As used herein, the articles “a” and “an” refer to one or to more thanone, i.e., to at least one, of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

The term “about,” as used herein, means approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20%.

“Addictive disorders” include, but are not limited to, eating disorders,obesity-related disorders, impulse control disorders, alcohol-relateddisorders, nicotine-related disorders, amphetamine-related disorders,methamphetamine-related disorders, cannabis-related disorders,cocaine-related disorders, gambling, sexual disorders, hallucinogen usedisorders, inhalant-related disorders, benzodiazepine abuse ordependence related disorders, and opioid-related disorders.

One of ordinary skill in the art will appreciate that addictivedisorders such as those related to alcohol or drugs, does mean that asubject is dependent unless specifically defined as such.

The term “additional therapeutically active compound”, in the context ofthe present invention, refers to the use or administration of a compoundfor an additional therapeutic use other than just the particulardisorder being treated. Such a compound, for example, could include onebeing used to treat an unrelated disease or disorder, or a disease ordisorder which may not be responsive to the primary treatment for theaddictive disease or disorder being treated. Disease and disorders beingtreated by the additional therapeutically active agent include, forexample, hypertension and diabetes.

As used herein, the term “aerosol” refers to suspension in the air. Inparticular, aerosol refers to the particlization or atomization of aformulation of the invention and its suspension in the air.

As used herein, the term “affected cell” refers to a cell of a subjectafflicted with a disease or disorder, which affected cell has an alteredphenotype compared with a subject not afflicted with a disease,condition, or disorder.

Cells or tissue are “affected” by a disease or disorder if the cells ortissue have an altered phenotype relative to the same cells or tissue ina subject not afflicted with a disease, condition, or disorder.

As used herein, an “agonist” is a composition of matter that, whenadministered to a mammal such as a human, enhances or extends abiological activity of interest. Such effect may be direct or indirect.

The term “alcohol abuser”, as used herein, refers to a subject who meetsDSM IV criteria for alcohol abuse (i.e., “repeated use despite recurrentadverse consequences”) but is not dependent on alcohol.

“Alcohol-related disorders” as used herein refers to diseases anddisorder related to alcohol consumption and include, but are not limitedto, alcohol-induced psychotic disorder, with delusions; alcohol abuse;excessive drinking; heavy drinking; problem drinking; alcoholintoxication; alcohol withdrawal; alcohol intoxication delirium; alcoholwithdrawal delirium; alcohol-induced persisting dementia;alcohol-induced persisting amnestic disorder; alcohol dependence;alcohol-induced psychotic disorder, with hallucinations; alcohol-inducedmood disorder; alcohol-induced or associated bipolar disorder;alcohol-induced or associated post traumatic stress disorder;alcohol-induced anxiety disorder; alcohol-induced sexual dysfunction;alcohol-induced sleep disorder; and alcohol-related disorder nototherwise specified (NOS).

As used herein, “amino acids” are represented by the full name thereof,by the three letter code corresponding thereto, or by the one-lettercode corresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

The expression “amino acid” as used herein is meant to include bothnatural and synthetic amino acids, and both D and L amino acids.“Standard amino acid” means any of the twenty standard L-amino acidscommonly found in naturally occurring peptides. “Nonstandard amino acidresidue” means any amino acid, other than the standard amino acids,regardless of whether it is prepared synthetically or derived from anatural source. As used herein, “synthetic amino acid” also encompasseschemically modified amino acids, including but not limited to salts,amino acid derivatives (such as amides), and substitutions. Amino acidscontained within the peptides of the present invention, and particularlyat the carboxy- or amino-terminus, can be modified by methylation,amidation, acetylation or substitution with other chemical groups whichcan change the peptide's circulating half-life without adverselyaffecting their activity. Additionally, a disulfide linkage may bepresent or absent in the peptides of the invention.

The term “amino acid” is used interchangeably with “amino acid residue,”and may refer to a free amino acid and to an amino acid residue of apeptide. It will be apparent from the context in which the term is usedwhether it refers to a free amino acid or a residue of a peptide.

Amino acids have the following general structure:

Amino acids may be classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains; (2) side chains containing ahydroxylic (OH) group; (3) side chains containing sulfur atoms; (4) sidechains containing an acidic or amide group; (5) side chains containing abasic group; (6) side chains containing an aromatic ring; and (7)proline, an imino acid in which the side chain is fused to the aminogroup.

As used herein, the term “conservative amino acid substitution” isdefined herein as exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides:

-   -   Asp, Asn, Glu, Gln;

III. Polar, positively charged residues:

-   -   His, Arg, Lys;

IV. Large, aliphatic, nonpolar residues:

-   -   Met Leu, Ile, Val, Cys

V. Large, aromatic residues:

-   -   Phe, Tyr, Trp

The nomenclature used to describe the peptide compounds of the presentinvention follows the conventional practice wherein the amino group ispresented to the left and the carboxy group to the right of each aminoacid residue. In the formulae representing selected specific embodimentsof the present invention, the amino- and carboxy-terminal groups,although not specifically shown, will be understood to be in the formthey would assume at physiologic pH values, unless otherwise specified.

The term “basic” or “positively charged” amino acid, as used herein,refers to amino acids in which the R groups have a net positive chargeat pH 7.0, and include, but are not limited to, the standard amino acidslysine, arginine, and histidine.

As used herein, an “analog” of a chemical compound is a compound that,by way of example, resembles another in structure but is not necessarilyan isomer (e.g., 5-fluorouracil is an analog of thymine).

An “antagonist” is a composition of matter that when administered to amammal such as a human, inhibits or impedes a biological activityattributable to the level or presence of an endogenous compound in themammal. Such effect may be direct or indirect.

As used herein, the term “anti-alcohol agent” refers to any active drug,formulation, or method that exhibits activity to treat or prevent one ormore symptom(s) of alcohol addiction, alcohol abuse, alcoholintoxication, and/or alcohol withdrawal, including drugs, formulationsand methods that significantly reduce, limit, or prevent alcoholconsumption in mammalian subjects.

The term “appetite suppression”, as used herein, is a reduction, adecrease or, in cases of excessive food consumption, an amelioration inappetite. This suppression reduces the desire or craving for food.Appetite suppression can result in weight loss or weight control asdesired.

The term “average drinking,” as used herein, refers to the mean numberof drinks consumed during a one week period. The term “average drinking”is used interchangeably herein with the term “average level ofdrinking.”

A “biomarker” is a specific biochemical in the body which has aparticular molecular feature that makes it useful for measuring theprogress of disease or the effects of treatment, or for measuring aprocess of interest.

A “compound,” as used herein, refers to any type of substance or agentthat is commonly considered a drug, or a candidate for use as a drug, aswell as combinations and mixtures of the above.

A “control” subject is a subject having the same characteristics as atest subject, such as a similar type of dependence, etc. The controlsubject may, for example, be examined at precisely or nearly the sametime the test subject is being treated or examined. The control subjectmay also, for example, be examined at a time distant from the time atwhich the test subject is examined, and the results of the examinationof the control subject may be recorded so that the recorded results maybe compared with results obtained by examination of a test subject.

A “test” subject is a subject being treated.

As used herein, a “derivative” of a compound refers to a chemicalcompound that may be produced from another compound of similar structurein one or more steps, as in replacement of H by an alkyl, acyl, or aminogroup.

As used herein, the term “diagnosis” refers to detecting a risk orpropensity to an addictive related disease disorder. In any method ofdiagnosis exist false positives and false negatives. Any one method ofdiagnosis does not provide 100% accuracy.

A “disease” is a state of health of a subject wherein the subject cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe subject's health continues to deteriorate. In contrast, a “disorder”in a subject is a state of health in which the subject is able tomaintain homeostasis, but in which the subject's state of health is lessfavorable than it would be in the absence of the disorder. However, thedefinitions of “disease” and “disorder” as described above are not meantto supersede the definitions or common usage related to specificaddictive diseases or disorders.

A disease, condition, or disorder is “alleviated” if the severity of asymptom of the disease or disorder, the frequency with which such asymptom is experienced by a patient, or both, are reduced.

As used herein, an “effective amount” means an amount sufficient toproduce a selected effect, such as alleviating symptoms of a disease ordisorder. In the context of administering two or more compounds, theamount of each compound, when administered in combination with anothercompound(s), may be different from when that compound is administeredalone. The term “more effective” means that the selected effect isalleviated to a greater extent by one treatment relative to the secondtreatment to which it is being compared.

The term “elixir,” as used herein, refers in general to a clear,sweetened, alcohol-containing, usually hydroalcoholic liquid containingflavoring substances and sometimes active medicinal agents.

“Ethnic and Racial Categories” are defined herein according to NIHguidelines (1997 OMB Directive 15).

Ethnic Categories:

Hispanic or Latino: A person of Cuban, Mexican, Puerto Rican, South orCentral American, or other Spanish culture or origin, regardless ofrace. The term “Spanish origin” can also be used in addition to“Hispanic or Latino.”

Not Hispanic or Latino

Racial Categories:

American Indian or Alaska Native: A person having origins in any of theoriginal peoples of North, Central, or South America, and who maintainstribal affiliations or community attachment.

Asian: A person having origins in any of the original peoples of the FarEast, Southeast Asia, or the Indian subcontinent including, for example,Cambodia, China, India, Japan, Korea, Malaysia, Pakistan, the PhilippineIslands, Thailand, and Vietnam. (Note: Individuals from the PhilippineIslands have been recorded as Pacific Islanders in previous datacollection strategies.)

Black or African American: A person having origins in any of the blackracial groups of Africa. Terms such as “Haitian” or “Negro” can be usedin addition to “Black or African American.”

Native Hawaiian or Other Pacific Islander: A person having origins inany of the original peoples of Hawaii, Guam, Samoa, or other PacificIslands.

White: A person having origins in any of the original peoples of Europe,the Middle East, or North Africa.

The term “excessive drinker,” as used herein, refers to men who drinkmore than 21 alcohol units per week and women who consume more than 14alcohol units per week. One standard drink is 0.5 oz of absolutealcohol, equivalent to 10 oz of beer, 4 oz of wine, or 1 oz of 100-proofliquor. These individuals are not dependent on alcohol but may or maynot meet DSM IV criteria for alcohol abuse.

As used herein, a “functional” molecule is a molecule in a form in whichit exhibits a property or activity by which it is characterized. Afunctional enzyme, for example, is one that exhibits the characteristiccatalytic activity by which the enzyme is characterized.

The term “heavy drinker,” as used herein, refers to men who drink morethan 14 alcohol units per week and women who consume more than 7 alcoholunits per week. One standard drink is 0.5 oz of absolute alcohol,equivalent to 10 oz of beer, 4 oz of wine, or 1 oz of 100-proof liquor.These individuals are not dependent on alcohol but may or may not meetDSM IV criteria for alcohol abuse.

The term “heavy drinking”, as used with respect to the alcohol-dependentpopulation of Example 1, refers to drinking at least 21 standarddrinks/week for women and at least 30 drinks/week for men during the 90days prior to enrollment in the study and is more fully describedtherein.

A “heavy drinking day,” as used herein, refers to the consumption by aman or woman of more than about five or four standard drinks perdrinking day, respectively.

The term “heavy drug use,” as used herein, refers to the use of any drugof abuse, including, but not limited to, cocaine, methamphetamine, otherstimulants, phencyclidine, other hallucinogens, marijuana, sedatives,tranquilizers, hypnotics, opiates at intervals or in quantities greaterthan the norm. The intervals of use include intervals such as at leastonce a month, at least once a week, and at least once a day. “Heavy druguse” is defined as testing “positive” for the use of that drug on atleast 2 occasions in any given week with at least 2 days between testingoccasions.

As used herein, the term “inhaler” refers both to devices for nasal andpulmonary administration of a drug, e.g., in solution, powder and thelike. For example, the term “inhaler” is intended to encompass apropellant driven inhaler, such as is used to administer antihistaminefor acute asthma attacks, and plastic spray bottles, such as are used toadminister decongestants.

The term “inhibit,” as used herein, refers to the ability of a compoundor any agent to reduce or impede a described function, level, activity,synthesis, release, binding, etc., based on the context in which theterm “inhibit” is used. Preferably, inhibition is by at least 10%, morepreferably by at least 25%, even more preferably by at least 50%, andmost preferably, the function is inhibited by at least 75%. The term“inhibit” is used interchangeably with “reduce” and “block.”

The term “inhibit a complex,” as used herein, refers to inhibiting theformation of a complex or interaction of two or more proteins, as wellas inhibiting the function or activity of the complex. The term alsoencompasses disrupting a formed complex. However, the term does notimply that each and every one of these functions must be inhibited atthe same time.

The term “inhibit a protein,” as used herein, refers to any method ortechnique which inhibits protein synthesis, levels, activity, orfunction, as well as methods of inhibiting the induction or stimulationof synthesis, levels, activity, or function of the protein of interest.The term also refers to any metabolic or regulatory pathway which canregulate the synthesis, levels, activity, or function of the protein ofinterest. The term includes binding with other molecules and complexformation. Therefore, the term “protein inhibitor” refers to any agentor compound, the application of which results in the inhibition ofprotein function or protein pathway function. However, the term does notimply that each and every one of these functions must be inhibited atthe same time.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of a compound of the invention in thekit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of alleviating the diseases ordisorders in a subject. The instructional material of the kit of theinvention may, for example, be affixed to a container which contains theidentified compound invention or be shipped together with a containerwhich contains the identified compound. Alternatively, the instructionalmaterial may be shipped separately from the container with the intentionthat the instructional material and the compound be used cooperativelyby the recipient.

“Intensity of drinking” refers to the number of drinks, which can beequated with values such as drinks/day, drinks/drinking day, etc.Therefore, greater intensity of drinking means more drinks/day, ordrinks/drinking day, etc.

As used herein, a “ligand” is a compound that specifically binds to atarget compound or molecule. A ligand “specifically binds to” or “isspecifically reactive with” a compound when the ligand functions in abinding reaction which is determinative of the presence of the compoundin a sample of heterogeneous compounds.

A “receptor” is a compound or molecule that specifically binds to aligand.

As used herein, the term “linkage” refers to a connection between twogroups. The connection can be either covalent or non-covalent, includingbut not limited to ionic bonds, hydrogen bonding, andhydrophobic/hydrophilic interactions.

As used herein, the term “linker” refers to a molecule that joins twoother molecules either covalently or noncovalently, e.g., through ionicor hydrogen bonds or van der Waals interactions.

The term “measuring the level of expression” or “determining the levelof expression” as used herein refers to any measure or assay which canbe used to correlate the results of the assay with the level ofexpression of a gene or protein of interest. Such assays includemeasuring the level of mRNA, protein levels, etc. and can be performedby assays such as northern and western blot analyses, binding assays,immunoblots, etc. The level of expression can include rates ofexpression and can be measured in terms of the actual amount of an mRNAor protein present.

The term “nasal administration” in all its grammatical forms refers toadministration of at least one compound of the invention through thenasal mucous membrane to the bloodstream for systemic delivery of atleast one compound of the invention. The advantages of nasaladministration for delivery are that it does not require injection usinga syringe and needle, it avoids necrosis that can accompanyintramuscular administration of drugs, and trans-mucosal administrationof a drug is highly amenable to self administration.

As used herein, the term “nucleic acid” encompasses RNA as well assingle and double-stranded DNA and cDNA. Furthermore, the terms,“nucleic acid,” “DNA,” “RNA” and similar terms also include nucleic acidanalogs, i.e. analogs having other than a phosphodiester backbone. Forexample, the so-called “peptide nucleic acids,” which are known in theart and have peptide bonds instead of phosphodiester bonds in thebackbone, are considered within the scope of the present invention. By“nucleic acid” is also meant any nucleic acid, whether composed ofdeoxyribonucleosides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages such as phosphodiester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridgedmethylene phosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, bridged phosphorothioate or sulfone linkages, andcombinations of such linkages. The term nucleic acid also specificallyincludes nucleic acids composed of bases other than the fivebiologically occurring bases (adenine, guanine, thymine, cytosine anduracil). Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction. Thedirection of 5′ to 3′ addition of nucleotides to nascent RNA,transcripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences”; sequences onthe DNA strand which are 3′ to a reference point on the DNA are referredto as “downstream sequences.”

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

“Obesity” is commonly referred to as a condition of increased bodyweight due to excessive fat. Drugs to treat obesity are generallydivided into three groups: (1) those that decrease food intake, such asdrugs that interfere with monoamine receptors, such as noradrenergicreceptors, serotonin receptors, dopamine receptors, and histaminereceptors; (2) those that increase metabolism; and (3) those thatincrease thermogenesis or decrease fat absorption by inhibitingpancreatic lipase (Bray, 2000, Nutrition, 16:953-960 and Leonhardt etal., 1999, Eur. J. Nutr., 38:1-13). Obesity has been defined in terms ofbody mass index (BMI). BMI is calculated as weight (kg)/[height (m)]²,according to the guidelines of the U.S. Centers for Disease Control andPrevention (CDC), and the World Health Organization (WHO). Physicalstatus: The use and interpretation of anthropometry. Geneva,Switzerland: World Health Organization 1995. WHO Technical ReportSeries), for adults over 20 years old, BMI falls into one of thesecategories: below 18.5 is considered underweight, 18.5-24.9 isconsidered normal, 25.0-29.9 is considered overweight, and 30.0 andabove is considered obese.

The term “oligonucleotide” typically refers to short polynucleotides,generally no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

The term “peptide” typically refers to short polypeptides.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.

The term “protein” typically refers to large polypeptides.

A “recombinant polypeptide” is one which is produced upon expression ofa recombinant polynucleotide.

A peptide encompasses a sequence of two or more amino acids wherein theamino acids are naturally occurring or synthetic (non-naturallyoccurring) amino acids. Peptide mimetics include peptides having one ormore of the following modifications:

1. peptides wherein one or more of the peptidyl —C(O)NR— linkages(bonds) have been replaced by a non-peptidyl linkage such as a—CH2-carbamate linkage (—CH2OC(O)NR—), a phosphonate linkage, a—CH2-sulfonamide (—CH2-S(O)2NR—) linkage, a urea (—NHC(O)NH—) linkage, a—CH2-secondary amine linkage, or with an alkylated peptidyl linkage(—C(O)NR—) wherein R is C1-C4 alkyl;

2. peptides wherein the N-terminus is derivatized to a —NRR1 group, to a—NRC(O)R group, to a —NRC(O)OR group, to a —NRS(O)2R group, to a—NHC(O)NHR group where R and R1 are hydrogen or C1-C4 alkyl with theproviso that R and R1 are not both hydrogen;

3. peptides wherein the C terminus is derivatized to —C(O)R2 where R2 isselected from the group consisting of C1-C4 alkoxy, and —NR3R4 where R3and R4 are independently selected from the group consisting of hydrogenand C1-C4 alkyl.

The term “per application” as used herein refers to administration of adrug or compound to a subject.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, and whichis not deleterious to the subject to which the composition is to beadministered.

A “predisposition” to an addictive disease or disorder refers tosituations a subject has an increased chance of abusing a substance suchas alcohol or a drug or becoming addicted to alcohol or a drug or otheraddictive diseases or disorders.

The term “prevent,” as used herein, means to stop something fromhappening, or taking advance measures against something possible orprobable from happening. In the context of medicine, “prevention”generally refers to action taken to decrease the chance of getting adisease or condition.

The term “problem drinker,” as used herein, encompasses individuals whodrink excessively and who report that their alcohol consumption iscausing them problems. Such problems include, for example, driving whileintoxicated, problems at work caused by excessive drinking, andrelationship problems caused by excessive drinking by the subject.

As used herein, “protecting group” with respect to a terminal aminogroup refers to a terminal amino group of a peptide, which terminalamino group is coupled with any of various amino-terminal protectinggroups traditionally employed in peptide synthesis. Such protectinggroups include, for example, acyl protecting groups such as formyl,acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl;aromatic urethane protecting groups such as benzyloxycarbonyl; andaliphatic urethane protecting groups, for example, tert-butoxycarbonylor adamantyloxycarbonyl. See Gross and Mienhofer, eds., The Peptides,vol. 3, pp. 3-88 (Academic Press, New York, 1981) for suitableprotecting groups.

As used herein, “protecting group” with respect to a terminal carboxygroup refers to a terminal carboxyl group of a peptide, which terminalcarboxyl group is coupled with any of various carboxyl-terminalprotecting groups. Such protecting groups include, for example,tert-butyl, benzyl, or other acceptable groups linked to the terminalcarboxyl group through an ester or ether bond.

The term “psychosocial management program,” as used herein, relates tothe use of various types of counseling and management techniques used tosupplement the combination pharmacotherapy treatment of addictive andalcohol-related diseases and disorders.

As used herein, the term “purified” and like terms relate to anenrichment of a molecule or compound relative to other componentsnormally associated with the molecule or compound in a nativeenvironment. The term “purified” does not necessarily indicate thatcomplete purity of the particular molecule has been achieved during theprocess. A “highly purified” compound as used herein refers to acompound that is greater than 90% pure.

“Reduce”—see “inhibit”.

The term “reduction in drinking”, as used herein, refers to a decreasein drinking according to one or more of the measurements of drinkingsuch as heavy drinking, number of drinks/day, number of drinks/drinkingday, etc.

The term “regulate” refers to either stimulating or inhibiting afunction or activity of interest.

A “sample,” as used herein, refers to a biological sample from asubject, including, but not limited to, normal tissue samples, diseasedtissue samples, biopsies, blood, saliva, feces, semen, tears, and urine.A sample can also be any other source of material obtained from asubject which contains cells, tissues, or fluid of interest asinterpreted in the context of the claim and the type of assay to beperformed using that sample.

By “small interfering RNAs (siRNAs)” is meant, inter alfa, an isolateddsRNA molecule comprising both a sense and an anti-sense strand. In oneaspect, it is greater than 10 nucleotides in length. siRNA also refersto a single transcript that has both the sense and complementaryantisense sequences from the target gene, e.g., a hairpin. siRNA furtherincludes any form of dsRNA (proteolytically cleaved products of largerdsRNA, partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA) as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution, and/oralteration of one or more nucleotides.

By the term “specifically binds,” as used herein, is meant a moleculewhich recognizes and binds a specific molecule, but does notsubstantially recognize or bind other molecules in a sample, or it meansbinding between two or more molecules as in part of a cellularregulatory process, where said molecules do not substantially recognizeor bind other molecules in a sample.

The term “standard,” as used herein, refers to something used forcomparison. For example, it can be a known standard agent or compoundwhich is administered or added and used for comparing results whenadding a test compound, or it can be a standard parameter or functionwhich is measured to obtain a control value when measuring an effect ofan agent or compound on a parameter or function. Standard can also referto an “internal standard”, such as an agent or compound which is addedat known amounts to a sample and is useful in determining such things aspurification or recovery rates when a sample is processed or subjectedto purification or extraction procedures before a marker of interest ismeasured. Internal standards are often a purified marker of interestwhich has been labeled, such as with a radioactive isotope, allowing itto be distinguished from an endogenous marker.

The term “one standard drink,” as used herein, is 0.5 oz of absolutealcohol, equivalent to 10 oz of beer, 4 oz of wine, or 1 oz of 100-proofliquor.

A “subject” of diagnosis or treatment is a mammal, including a human.

The term “subject comprises a predisposition to the early onset ofalcoholism,” as used herein, refers to a subject who has, or ischaracterized by, a predisposition to the early onset of alcoholism.

The term “symptom,” as used herein, refers to any morbid phenomenon ordeparture from the normal in structure, function, or sensation,experienced by the patient and indicative of disease. In contrast, asign is objective evidence of disease. For example, a bloody nose is asign. It is evident to the patient, doctor, nurse and other observers.

As used herein, term “treating” may include prophylaxis of the specificdisease, disorder, or condition, or alleviation of the symptomsassociated with a specific disease, disorder or condition and/orpreventing or eliminating said symptoms. A “prophylactic” treatment is atreatment administered to a subject who does not exhibit signs of adisease or exhibits only early signs of the disease for the purpose ofdecreasing the risk of developing pathology associated with the disease.“Treating” is used interchangeably with “treatment” herein.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

A “therapeutically effective amount” of a compound is that amount ofcompound which is sufficient to provide a beneficial effect to thesubject to which the compound is administered.

Chemical Definitions

As used herein, the term “halogen” or “halo” includes bromo, chloro,fluoro, and iodo.

The term “haloalkyl” as used herein refers to an alkyl radical bearingat least one halogen substituent, for example, chloromethyl, fluoroethylor trifluoromethyl and the like.

The term “C₁-C_(n) alkyl” wherein n is an integer, as used herein,represents a branched or linear alkyl group having from one to thespecified number of carbon atoms. Typically, C₁-C₆ alkyl groups include,but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl,iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl, and the like.

The term “C₂-C_(n) alkenyl” wherein n is an integer, as used herein,represents an olefinically unsaturated branched or linear group havingfrom two to the specified number of carbon atoms and at least one doublebond. Examples of such groups include, but are not limited to,1-propenyl, 2-propenyl, 1,3-butadienyl, 1-butenyl, hexenyl, pentenyl,and the like.

The term “C₂-C_(n) alkynyl” wherein n is an integer refers to anunsaturated branched or linear group having from two to the specifiednumber of carbon atoms and at least one triple bond. Examples of suchgroups include, but are not limited to, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 1-pentynyl, and the like.

The term “C₃-C_(n) cycloalkyl” wherein n=8, represents cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

As used herein, the term “optionally substituted” refers to from zero tofour substituents, wherein the substituents are each independentlyselected. Each of the independently selected substituents may be thesame or different than other substituents.

As used herein the term “aryl” refers to an optionally substituted mono-or bicyclic carbocyclic ring system having one or two aromatic ringsincluding, but not limited to, phenyl, benzyl, naphthyl,tetrahydronaphthyl, indanyl, indenyl, and the like. “Optionallysubstituted aryl” includes aryl compounds having from zero to foursubstituents, and “substituted aryl” includes aryl compounds having oneor more substituents. The term (C₅-C₈ alkyl)aryl refers to any arylgroup which is attached to the parent moiety via the alkyl group.

The term “heterocyclic group” refers to an optionally substituted mono-or bicyclic carbocyclic ring system containing from one to threeheteroatoms wherein the heteroatoms are selected from the groupconsisting of oxygen, sulfur, and nitrogen. As used herein the term“heteroaryl” refers to an optionally substituted mono- or bicycliccarbocyclic ring system having one or two aromatic rings containing fromone to three heteroatoms and includes, but is not limited to, furyl,thienyl, pyridyl and the like.

The term “bicyclic” represents either an unsaturated or saturated stable7- to 12-membered bridged or fused bicyclic carbon ring. The bicyclicring may be attached at any carbon atom which affords a stablestructure. The term includes, but is not limited to, naphthyl,dicyclohexyl, dicyclohexenyl, and the like.

The compounds of the present invention contain one or more asymmetriccenters in the molecule. In accordance with the present invention astructure that does not designate the stereochemistry is to beunderstood as embracing all the various optical isomers, as well asracemic mixtures thereof.

The compounds of the present invention may exist in tautomeric forms andthe invention includes both mixtures and separate individual tautomers.For example the following structure:

is understood to represent a mixture of the structures:

The term “pharmaceutically-acceptable salt” refers to salts which retainthe biological effectiveness and properties of the compounds of thepresent invention and which are not biologically or otherwiseundesirable. In many cases, the compounds of the present invention arecapable of forming acid and/or base salts by virtue of the presence ofamino and/or carboxyl groups or groups similar thereto.

Embodiments

The number of serotonin transporter protein molecules in cells isaffected by the amount of mature (secondary) serotonin transporter mRNAmolecules expressed in that cell. The expression levels of mRNA arecontrolled by the 5′-HTTLPR and 3′-UTR of the SLC6A4 gene via twodifferent mechanisms. The 5′-HTTLPR region controls the transcriptionrate of SLC6A4 (Heils et al., (1996) J. Neurochem. 66:2621-2624), whilers1042173 SNP in the 3′-UTR, of SLC6A4 affects mature mRNA levels viapost-transcriptional mechanisms (Battersby et al., (1999), J. Neurochem.72:1384-1388; Beaudoing et al., (2000), Genome Res 10:1001-1010; Chen etal., (2006), Nat. Genet. 38:1452-145).

The 5′-HTTLPR is found to harbor several binding sites for differenttranscription factor molecules necessary for the regulation oftranscription initiation (Hu et al., (2005), Alcohol Clin. Exp. Res.29:8-16). Therefore, the number of nascent (primary) mRNA copiestranscribed by the SLC6A4 gene and the subsequent mature mRNA copies isaffected by 5′-HTTLPR polymorphisms. The rs1042173 allelic differencesare reported to be regulated by Micro RNA (miRNA) binding to at/near thers1042173 site (for examples, miR-15a and miR-16 binding), degradingprimary mRNA molecules and differential polyadenylation and resulting inaltered mature mRNA levels. Therefore, the combined effects of 5′-HTTLPRand rs1042173 polymorphisms may modulate each other's individual effectson determining the overall availability of mature mRNA for translationinto serotonin transporter protein molecules.

Without wishing to be bound by any particularly, it is hypothesizedherein that, considering these factors, the combined genetic effect of5′-HTTLPR and rs1042173 polymorphisms can result in differences inserotonergic function and regulation. Since alcohol consumption affectsserotonergic function, this gene-gene interaction (5′-HTTLPR andrs1042173) may lead to a serotonergic dysregulation that eitherprovokes, aggravates, or maintains further drinking behavior andalcoholism. These states of serotonergic dysregulation or alterations infunction can be stabilized or ameliorated in excessive drinking oralcoholic populations by the compositions and methods of the presentinvention such as administration of serotonergic medications includingthe serotonin-3 (5-HT-3) antagonist, ondansetron.

In one embodiment, 5-HT-3 receptor antagonists (including ondansetron)can improve the drinking outcomes of those with certain polymorphisms of5′-HTTLPR and/or rs1042173, either alone or combined. Because abuseddrugs are predicted to work through similar mechanisms, the presentinvention therefore encompasses the use of 5-HT3 antagonists (includingondansetron) to ameliorate or stabilize these serotonergic states andproduce a therapeutic effect that improves clinical outcome for thesedisorders and diseases. The addictive diseases and disorders encompassedby the present compositions and methods include, but are not limited to,alcohol-related diseases and disorders, obesity-related diseases anddisorders, eating disorders, impulse control disorders, nicotine-relateddisorders, amphetamine-related disorders, methamphetamine-relateddisorders, cannabis-related disorders, cocaine-related disorders,hallucinogen use disorders, inhalant-related disorders, benzodiazepineabuse or dependence related disorders, opioid-related disorders,gambling, and computer or electronic addictions.

Because the serotonin system has intimate connections and is modulatedin the brain by other neurotransmitters, particularly dopamine, GABA,glutamate, opioids, and cannabinoid, the present invention encompassesthe use of medications and drugs that affect the structure and functionof these other neurotransmitters when combined with any serotonergicagent (including ondansetron). In one aspect, the combination isefficacious for individuals with polymorphisms at the 5′-HTTLPR andrs1042173 described herein or anywhere else in the serotonergic system.In another aspect, the present invention provides compositions,compounds and methods that are associated with these co-modulatingneurotransmitters (i.e., dopamine, GABA, glutamate, opioids, andcannabinoid), including, but not limited to, topiramate, baclofen,gabapentin, naltrexone, nalmefene, and rimonabant—in combination withany serotonergic agent (including but not limited to ondansetron,selective serotonin re-uptake blockers, and other agonists orantagonists of other serotonin receptors or moieties) can produce atherapeutic effect to improve the clinical outcomes for individuals whouse, abuse, misuse, or are dependent on alcohol. Because abused drugsare predicted to work through similar mechanisms, the present inventionfurther provides combinations of these co-modulating drugs with anyother serotonergic agent to be used to treat individuals with anysubstance use, abuse, misuse, dependence, or habit-forming behavior withpolymorphisms at 5′-HTTLPR and rs1042173, or anywhere else in theserotonergic or co-modulating neurotransmitter systems (i.e., dopamine,GABA, glutamate, opioids, and cannabinoid), either alone or incombination.

The present invention encompasses compositions and methods for treatmentor prevention where 5′-HTTLPR polymorphisms are associated withvulnerability or can sustain, provoke, or govern alcohol consumption.5′-HTTLPR polymorphisms or related miRNA, mRNA, protein expression,levels, or states of function, or other biochemical products or chemicalassociations may themselves serve as a biomarker for alcoholconsumption. Such a biomarker (i.e., blood test) can be used to providea means or a test to determine whether, and how much, alcohol has beenconsumed by an individual. 5′-HTTLPR polymorphisms or related miRNA,mRNA, protein expression, levels, or states of function, or otherbiochemical products or chemical associations may themselves serve as abiomarker for alcohol use, misuse, or dependence. Such a biomarker(i.e., blood test) can be used to provide a means or a test todetermine, evaluate, or support a diagnosis of alcohol use, misuse, ordependence.

The present invention encompasses compositions and methods for treatmentor prevention where rs1042173 polymorphisms are associated withvulnerability or can sustain, provoke, or govern alcohol consumption.rs1042173 polymorphisms or related miRNA, mRNA, protein expression,levels, or states of function, or other biochemical products or chemicalassociations may themselves serve as a biomarker for alcoholconsumption. Such a biomarker (i.e., blood test) can be used to providea means or a test to determine whether, and how much, alcohol has beenconsumed by an individual. rs1042173 polymorphisms or related miRNA,mRNA, protein expression, levels, or states of function, or otherbiochemical products or chemical associations may themselves serve as abiomarker for alcohol use, misuse, or dependence. Such a biomarker(i.e., blood test) can be used to provide a means or a test todetermine, evaluate, or support a diagnosis of alcohol use, misuse, ordependence.

The present invention encompasses compositions and methods for treatmentor prevention where the combination of 5′-HTTLPR and rs1042173polymorphisms is associated with vulnerability or can sustain, provoke,or govern alcohol consumption. The combination of 5′-HTTLPR andrs1042173 polymorphisms or related miRNA, mRNA, or protein expression,levels, or states of function, or other biochemical products or chemicalassociations, may itself serve as a biomarker for alcohol consumption.Such a biomarker can be used to provide a means or a test to determinewhether, and how much, alcohol has been consumed by an individual. Thecombination of 5′-HTTLPR and rs1042173 polymorphisms or related miRNA,mRNA, or protein expression, levels, or states of function, or otherbiochemical products or chemical associations, may itself serve as abiomarker for alcohol use, abuse, or dependence. Such a biomarker can beused to determine, evaluate, or support a diagnosis of alcohol use,misuse, or dependence.

The present invention further provides for the use of any 5-HT-3antagonist (including ondansetron) at any dose or dosage form toindividuals with 5′-HTTLPR polymorphisms can ameliorate, improve, treat,or aid in recovery from alcohol use, abuse, or dependence, or substanceuse, abuse, or dependence.

The present invention encompasses providing any agent or drug that hasan effect on the serotonin system, at any dose or dosage form, eitherdirectly or indirectly to individuals with 5′-HTTLPR polymorphisms canameliorate, improve, treat, or aid in recovery from alcohol use, abuse,or dependence.

The present invention encompasses providing any 5-HT-3 antagonist(including ondansetron), at any dose or any dosage form, to individualswith rs1042173 polymorphisms can ameliorate, improve, treat or aid inrecovery from alcohol use, abuse, or dependence, or substance use,abuse, or dependence.

The present invention encompasses providing any agent or drug that hasan effect on the serotonin system, at any dose or dosage thrill, eitherdirectly or indirectly to individuals with rs1042173 polymorphisms canameliorate, improve, treat, or aid in recovery from substance use,misuse, abuse, or dependence or any habit-forming behavior.

The present invention encompasses providing any 5-HT-3 antagonistincluding ondansetron), at any dose or dosage form, to individuals withrs1042173 and 5′-HTTLPR polymorphisms combined can ameliorate, improve,treat, or aid in recovery from alcohol use, abuse, or dependence.

The present invention encompasses providing any agent or drug that hasan effect on the serotonin system, at any dose or dosage form, eitherdirectly or indirectly to individuals with rs1042173 and 5′-HTTLPRpolymorphisms can ameliorate, improve, treat, or aid in recovery fromsubstance use, abuse, or dependence or habit-forming behavior.

The present invention encompasses providing any agent or drug orchemical entity that has an effect, at any dose or dosage form, eitherdirectly or indirectly to modulate, regulate, or alter the structural,functional, molecular, or biochemical effects of rs1042173 and 5′-HTTLPRpolymorphisms, either alone or combined, can ameliorate, improve, treat,or aid in recovery from alcohol use, abuse, or dependence.

The present invention encompasses providing any agent or drug orchemical entity that has an effect, at any dose or dosage form, eitherdirectly or indirectly to modulate, regulate, or alter the structural,functional, molecular, or biochemical effects of rs1042173 and 5′-HTTLPRpolymorphisms, either alone or combined, can ameliorate, improve, treat,or aid in recovery from substance use, abuse, or dependence orhabit-forming behavior.

5′-HTTLPR and rs1042173 polymorphisms, either alone or in combination,or combined with any other polymorphisms within the serotonin system, ortheir related miRNA, mRNA, ncRNA, or protein expression, levels, orstates of function or other biochemical products or chemicalassociations, may themselves serve as a biomarker for alcoholconsumption. In one aspect, the use of 5′-HTTLPR and rs1042173 asbiomarkers can provide a means or a test to determine whether, and howmuch, alcohol has been consumed by an individual.

5′-HTTLPR and rs1042173 polymorphisms, either alone or in combination,or combined with any other polymorphisms within the serotonin system, ortheir related miRNA, mRNA, ncRNA, or protein expressions, levels, orstates of function or other biochemical products or chemicalassociations, may themselves serve as a biomarker for alcohol use,misuse, or dependence. In one aspect, the use of 5′-HTTLPR and rs1042173as biomarkers can provide a means or a test to determine, evaluate, orsupport a diagnosis of alcohol use, misuse, abuse, or dependence.

5′-HTTLPR and rs1042173 polymorphisms, either alone or in combination,or combined with any other polymorphisms within the serotonin system, ortheir related miRNA, mRNA, ncRNA, or protein expressions, levels, orstates of function or other biochemical products or chemicalassociations, may themselves serve as a biomarker for substance use or ahabit-forming behavior. In one aspect, the use of 5′-HTTLPR andrs1042173 as biomarkers can provide a means or a test to determinewhether, and how much, substance has been consumed, or a habit-formingbehavior has been performed, by an individual.

5′-HTTLPR and rs1042173 polymorphisms, either alone or in combination,or combined with any other polymorphisms within the serotonin system, ortheir related miRNA, mRNA, ncRNA, or protein expressions, levels, orstates of function or other biochemical products or chemicalassociations, may themselves serve as a biomarker for substance use,abuse, misuse, dependence, or any habit-forming behavior. In one aspect,the use of 5′-HTTLPR and rs1042173 as biomarkers can provide a means ora test to determine whether, and how much, a substance has beenconsumed, or a habit-forming behavior has been performed, by anindividual.

In one aspect, providing any agent or drug or chemical entity, at anydose or dosage form, either directly or indirectly to modulate,regulate, or alter the structural, functional, molecular, or biochemicaleffects of the serotonin system can be used to predict the response of atreatment effect toward any genetic polymorphism, either alone orcombined, to ameliorate, improve, treat, or aid in recovery from alcoholuse, abuse, or dependence, substance use, abuse, or dependence, or anyhabit-forming behavior.

In another aspect, providing any 5-HT-3 antagonist (includingondansetron), at any dose or dosage form, either directly or indirectlyto modulate, regulate, or alter the structural, functional, molecular,or biochemical effects of any polymorphism, either alone or combined,within the serotonin system can ameliorate, improve, treat, or aid inrecovery from alcohol use, abuse, or dependence or substance use, abuse,or dependence

The present invention further encompasses a method whereby geneticscreening is used to identify polymorphisms within the serotonin systemor their related miRNA, mRNA, ncRNA, or protein expression, levels, orstates of function, or other biochemical products or chemicalassociations, may serve as a biomarker for alcohol consumption. Such abiomarker (including a blood test) can be used to provide a means or atest to determine whether, and how much, alcohol has been consumed by anindividual.

The present invention further encompasses a method whereby geneticscreening is used to identify polymorphisms within the serotonin systemor their related miRNA, mRNA, ncRNA, or protein expression, levels, orstates of function, or other biochemical products or chemicalassociations, may serve as a biomarker to determine, evaluate, orsupport the diagnosis of alcohol use, misuse, abuse, or dependence.

The present invention further encompasses a method whereby geneticscreening is used to identify polymorphisms within the serotonin systemor their related miRNA, mRNA, ncRNA, or protein expression, levels, orstates of function, or other biochemical products or chemicalassociations, may serve as a biomarker for the consumption of anysubstance, or any substance with abuse- or dependence-formingcapability. Such a biomarker (including a blood test) can be used toprovide a means or a test to determine whether, and how much of, anysubstance (including addictive substances) has been consumed, or ahabit-forming behavior has been performed, by an individual.

The present invention further encompasses a method whereby geneticscreening is used to identify polymorphisms within the serotonin systemor their related miRNA, mRNA, ncRNA, or protein expression, levels, orstates of function, or other biochemical products or chemicalassociations, may serve as a biomarker to determine, evaluate, orsupport the diagnosis of substance use, misuse, abuse, or dependence orany habit-forming behavior.

The present invention further encompasses a method whereby geneticscreening is used to identify polymorphisms within the serotonin systemor their related miRNA, mRNA, ncRNA, or protein expression, levels, orstates of function, or other biochemical products or chemicalassociations, in order to identify individuals who use, abuse, misuse,or are dependent on alcohol or any other substance or habit-formingbehavior, may be a basis for identifying treatment. Such a test(including a blood test) can be anticipated to determine individuals whowill respond to any treatment (i.e., pharmacological, behavioral,genetic, biochemical, or any other combinations).

The present invention further encompasses a method whereby geneticscreening is used to identify any polymorphisms within the serotoninsystem or their related miRNA, mRNA, ncRNA, or protein expressions,levels, or states of function, or other biochemical products or chemicalassociations, in order to identify individuals who use, abuse, misuse,or are dependent on any substance or have a habit-forming behavior, maybe a basis for identifying adverse events or side effects or optimizingany treatment (i.e., pharmacological, behavioral, genetic, biochemical,or any other combinations) at any dose, dosage form, or treatmentregimen. Such a test can be anticipated to determine individuals whowill not respond to a treatment or individuals who will need additionalmeasures to optimize the success of any treatment (i.e.,pharmacological, behavioral, genetic, biochemical, or any othercombinations).

The present invention further encompasses a method whereby geneticscreening is used to identify any polymorphisms within the serotoninsystem or their related miRNA, mRNA, ncRNA, or protein expression,levels, or states of function, or other biochemical products or chemicalassociations, in order to identify individuals who use, abuse, misuse,or are dependent on alcohol or any other substance or have ahabit-forming behavior (including but not limited to obesity, gambling,or computer or electronic addictions), may be a basis for identifyingadverse events or side effects or optimizing treatment with any 5-HT-3antagonist (including ondansetron) at any dose or dosage form. Such atest can be anticipated to determine individuals who will respond totreatment with any 5-HT-3 antagonist (including ondansetron).

The present invention further encompasses a method whereby geneticscreening is used to identify 5′-HTTLPR and rs1042173 polymorphisms ortheir related miRNA, mRNA, or protein expressions, levels, or states offunction, or other biochemical products or chemical associations, eitheralone or in any combination, in order to identify individuals who use,abuse, misuse, or are dependent on alcohol or any other substance orhave a habit-forming behavior, may be a basis for identifying adverseevents or side effects or optimizing treatment with any 5-HT-3antagonist (including ondansetron) at any dose or dosage form. Such atest (including a blood test) can be anticipated to determineindividuals who will not respond to treatment with a 5-HT-3 antagonist(including ondansetron) or individuals who will need additional measuresto optimize the success of treatment with any 5-HT-3 antagonist(including ondansetron).

The present invention further encompasses a method whereby geneticscreening is used to identify 5′-HTTLPR and rs1042173 polymorphisms ortheir related miRNA, mRNA, or protein expressions, levels, or states offunction, or other biochemical products or chemical associations, eitheralone or in any combination, in order to identify individuals who use,abuse, misuse, or are dependent on alcohol or any other substance orhave a habit-forming behavior (including but not limited to obesity,gambling, or computer or electronic addictions), may be a basis foridentifying those who will respond to treatment with any 5-HT-3antagonist (including ondansetron) at any dose or dosage form. Such atest (including a blood test) can be anticipated to determineindividuals who will respond to treatment with a 5-HT-3 antagonist(including ondansetron). Such diseased individuals can be identified bymeans of the genetic screening and then provided with ondansetron.

The present invention further encompasses a method whereby geneticscreening is used to identify 5′-HTTLPR and rs1042173 polymorphisms ortheir related miRNA, mRNA, or protein expressions, levels, or states offunction, or other biochemical products or chemical associations, eitheralone or in any combination, in order to identify individuals who use,abuse, misuse, or are dependent on alcohol, may be a basis foridentifying those who will respond to treatment with any 5-HT-3antagonist (including ondansetron) at any dose or dosage form. Such atest (including a blood test) can be anticipated to determineindividuals with an alcohol use, abuse, or dependence disorder who wiltrespond to treatment with a 5-HT-3 antagonist (including ondansetron).Such individuals can be identified by means of the genetic screening andthen provided with ondansetron.

The present invention further encompasses a method whereby geneticscreening is used to identify any polymorphisms within the serotoninsystem or their related miRNA, mRNA, ncRNA, or protein expressions,levels, or states of function, or other biochemical products or chemicalassociations, either alone or in any combination, in order to identifyindividuals who use, abuse, misuse, or are dependent on alcohol or anyother substance or have a habit-forming behavior (including but notlimited to obesity, gambling, or computer or electronic addictions), maybe a basis for identifying individuals who will respond to treatmentwith any serotonergic agent, compound, or drug at any dose or dosageform. Such a test (including a blood test) can be anticipated todetermine individuals who will respond to treatment for any addictivebehavior with any serotonergic agent, compound, or drug.

The present invention further encompasses a method whereby geneticscreening is used to identify 5′-HTTLPR and rs1042173 polymorphisms ortheir related miRNA, mRNA, or protein expressions, levels, or states offunction, or other biochemical products or chemical associations, eitheralone or in any combination, in order to identify individuals who use,abuse, misuse, or are dependent on alcohol or any other substance orhave a habit-forming behavior (including but not limited to obesity,gambling, or computer or electronic addictions), may be a basis foridentifying those susceptible to adverse events or side effects oroptimizing treatment with any serotonergic agent, compound, or drug atany dose or dosage form. Such a test (including a blood test) can beanticipated to determine individuals who will not respond to treatmentwith a serotonergic agent, compound, or drug, or individuals who willneed additional measures to optimize the success of treatment with anyserotonergic agent, compound, or drug.

The present invention further encompasses a method whereby geneticscreening is used to identify 5′-HTTLPR and rs1042173 polymorphisms, orany other polymorphisms in co-modulating neurotransmitter systems (i.e.,dopamine, GABA, glutamate, opioids, and cannabinoids) or their relatedmiRNA, mRNA, ncRNA, or protein expressions, levels, or states offunction, or other biochemical products or chemical associations, eitheralone or in any combination, in order to identify individuals who use,abuse, misuse, or are dependent on alcohol, may be a basis foridentifying those who will respond to treatment with any 5-HT-3antagonist (including ondansetron) in combination with any drug thataffects these co-modulating systems (including but not limited totopiramate, baclofen, gabapentin, naltrexone, nalmefene, and rimonabant)at any dose or dosage form. Such a test (including a blood test) can beanticipated to determine individuals with an alcohol use, abuse, ordependence disorder who will respond to treatment with a 5-HT-3antagonist (including ondansetron) plus any of these co-modulatingagents or drugs. Individuals with alcohol use, abuse, or dependence whohave these polymorphisms identified by this genetic screening can thanbe provided with ondansetron plus the co modulating medication or drug,with the prediction that these combinations will be expected to beefficacious.

The present invention further encompasses a method whereby geneticscreening is used to identify 5′-HTTLPR and rs1042173 polymorphisms, orany other polymorphisms in co-modulating neurotransmitter systems (i.e.,dopamine, GABA, glutamate, opioids, and cannabinoids) or their relatedmiRNA, mRNA, ncRNA, or protein expressions, levels, or states offunction, or other biochemical products or chemical associations, eitheralone or in any combination, in order to identify individuals who use,abuse, misuse, or are dependent on alcohol, may be a basis foridentifying those who will be susceptible to adverse events or notrespond to treatment with any 5-HT-3 antagonist (including ondansetron)in combination with any drug that affects these co-modulating systems(including but not limited to topiramate, baclofen, gabapentin,naltrexone, nalmefene, and rimonabant) at any dose or dosage form. Sucha test (including a blood test) can be anticipated to determineindividuals who will not respond to treatment with a 5-HT-3 antagonist(including ondansetron) plus any of these co-modulating agents or drugs,or who will need additional measures to optimize treatment to thesecompounds. Individuals with substance use, abuse, dependence or anyhabit-forming behavior who have these polymorphisms identified by thisgenetic screening can either be screened out of being provided thecombined treatment or be given additional measures to optimize theirtreatment.

The present invention further encompasses a method whereby geneticscreening is used to identify 5′-HTTLPR and rs1042173 polymorphisms, orany other polymorphisms in co-modulating neurotransmitter systems (i.e.,dopamine, GABA, glutamate, opioids, and cannabinoids) or their relatedmiRNA, mRNA, ncRNA, or protein expressions, levels, or states offunction, or other biochemical products or chemical associations, eitheralone or in any combination, is used to produce a biomarker (including ablood test) to determine, ascertain, or evaluate consumption level ordiagnosis of alcohol use, abuse, misuse, or dependence.

The present invention further encompasses a method whereby geneticscreening is used to identify 5′-HTTLPR and rs1042173 polymorphisms, orany other polymorphisms in co-modulating neurotransmitter systems (i.e.,dopamine, GABA, glutamate, opioids, and cannabinoids) or their relatedmiRNA, mRNA, ncRNA, or protein expressions, levels, or states offunction, or other biochemical products or chemical associations, eitheralone or in any combination, is used to produce a biomarker (including ablood test) to determine, ascertain, or evaluate consumption level ordiagnosis of substance use, abuse, misuse, or dependence orhabit-forming behavior.

The present invention further encompasses a method whereby geneticscreening is used to identify 5′-HTTLPR and rs1042173 polymorphisms, orany other polymorphisms in co-modulating neurotransmitter systems (i.e.,dopamine, GABA, glutamate, opioids, and cannabinoids) or their relatedmiRNA, mRNA, ncRNA, or protein expressions, levels, or states offunction, or other biochemical products or chemical associations, eitheralone or in any combination, in order to identify individuals withsubstance use, abuse, misuse, or dependence or any habit-formingbehavior, may be a basis for identifying those who will respond totreatment with any 5-HT-3 antagonist (including ondansetron) incombination with any drug that affects these co-modulating systems(including but not limited to topiramate, baclofen, gabapentin,naltrexone, nalmefene, and rimonabant) at any dose or dosage form. Sucha test (including a blood test) can be anticipated to determineindividuals with substance use, misuse, abuse, or dependence or anyhabit-forming disorder who will respond to treatment with a 5-HT-3antagonist (including ondansetron) plus any of these co-modulatingagents or drugs. Individuals with substance use, abuse, or dependence orany habit-forming behavior who have these polymorphisms identified bythis genetic screening can than be provided with ondansetron plus the comodulating medication or drug, with the prediction that thesecombinations will be expected to be efficacious.

The present invention further encompasses a method whereby geneticscreening is used to identify 5′-HTTLPR and rs1042173 polymorphisms, orany other polymorphisms in co-modulating neurotransmitter systems (i.e.,dopamine, GABA, glutamate, opioids, and cannabinoids) or their relatedmiRNA, mRNA, ncRNA, or protein expressions, levels, or states offunction, or other biochemical products or chemical associations, eitheralone or in any combination, in order to identify individuals withsubstance use, abuse, misuse, or dependence or any habit-formingbehavior, may be a basis for identifying those who will be susceptibleto adverse events or side effects or who will not respond to treatmentwith any 5-HT-3 antagonist (including ondansetron) in combination withany drug that affects these co-modulating systems (including but notlimited to topiramate, baclofen, gabapentin, naltrexone, nalmefene, andrimonabant) at any dose or dosage form. Such a test (including a bloodtest) can be anticipated to determine individuals with substance use,misuse, abuse, or dependence or any habit-forming disorder who will notrespond to treatment with a 5-HT-3 antagonist (including ondansetron)plus any of these co-modulating agents or drugs, or who will requireadditional measures to optimize treatment. Individuals with substanceuse, abuse, dependence, or any habit-forming behavior who have thesepolymorphisms identified by this genetic screening can either bescreened out of being provided the combined treatment or be givenadditional measures to optimize their treatment.

The present invention encompasses the use of ondansetron as well asother drugs. In one aspect, combinations of drugs are used. The presentinvention encompasses the use of combinations of drugs or compounds totreat addictive and compulsive diseases and disorders, particularalcohol-related diseases and disorders. The present invention furtherencompasses the use of adjunctive treatments and therapy such aspsychosocial management regimes, hypnosis, and acupuncture.

In one embodiment, the present invention provides compositions andmethods for treating alcohol-related diseases and disorders usingpharmaceutical compositions comprising effective amounts of ondansetron,topiramate and/or naltrexone.

The dosage of the active compound(s) being administered will depend onthe condition being treated, the particular compound, and other clinicalfactors such as age, sex, weight, and health of the subject beingtreated, the route of administration of the compound(s), and the type ofcomposition being administered (tablet, gel cap, capsule, solution,suspension, inhaler, aerosol, elixir, lozenge, injection, patch,ointment, cream, etc.). It is to be understood that the presentinvention has application for both human and veterinary use.

For example, in one embodiment relating to oral administration tohumans, a dosage of between approximately 0.1 and 300 mg/kg/day, orbetween approximately 0.5 and 50 mg/kg/day, or between approximately 1and 10 mg/kg/day, is generally sufficient, but will vary depending onsuch things as the disorder being treated, the length of treatment, theage, sex, weight, and/or health of the subject, etc. The drugs can beadministered in formulations that contain all drugs being used, or thedrugs can be administered separately. In some cases, it is anticipatedthat multiple doses/times of administration will be required or useful.The present invention further provides for varying the length of time oftreatment.

Topiramate is disclosed herein as a drug useful in combination drugtherapy. In one embodiment, topiramate is provided at a dosage rangingfrom about 15 mg/day to about 2500 mg/day. In one aspect, topiramate isadministered at a dosage ranging from about 25 mg/day to about 1000mg/day. In yet another aspect, topiramate is administered at a dosageranging from about 50 mg/day to about 500 mg/day. In one aspect,topiramate is administered at a dosage of about 400 mg/day. In anotheraspect, topiramate is administered at a dosage of 400 mg/day. In afurther aspect, topiramate is administered at a dosage of about 300mg/day. In yet a further aspect, topiramate is administered at a dosageof about 275 mg/day. In one aspect, topiramate is administered at a doseof about 1 mg/day. In one aspect, up to about 300 mg/day isadministered.

In one embodiment, topiramate is provided at a dose of about 1 mg/kg. Inone aspect, topiramate is provided at a dose of about 10 mg/kg. In oneaspect, topiramate is provided at a dose of about 100 mg/kg. In oneembodiment, topiramate is administered at a dosage ranging from about0.1 mg/kg/day to about 100 mg/kg/day.

Topiramate (C₁₂H₂₁NO₈S; IUPAC name:2,3:4,5-Bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate; CASRegistry No. 97240-79-4) has the following structure:

An important aspect of psychotropic drugs is to produce weight gain.These increases in weight gain can induce a range of metabolic problemsincluding abnormal sugar, fat, and carbohydrate metabolism. Becausetopiramate can cause weight loss and improve endocrine function, it isproposed herein that topiramate may be used to ameliorate weight gaincaused by other psychotropic drugs with which it is combined as well asalcohol and any other abused drugs.

An important adverse event of topiramate is cognitive impairment. In thegeneral population, this is reported by 2.4% of individuals who taketopiramate (Johnson & Johnson Pharmaceutical Research & Development.Investigator's Brochure: Topiramate (RWJ-17021-000), 10th ed.; December2005). In the substance abuse field, the occurrence rate of cognitiveimpairment is about 18.7% (Johnson B A, Ait-Daoud N, Bowden C L et al.Oral topiramate for treatment of alcohol dependence: a randomizedcontrolled trial. Lancet 2003, 361:1677-1685). Topiramate-associatedcognitive effects are due to its anti-glutaminergic properties. It is,therefore, not obvious that ondansetron, a serotonin-3 receptorantagonist, will alleviate these complaints of cognitive impairment.Ondansetron appears to have cholinergic effects, perhaps thoughinteractions with the GABA system, that seem to amelioratetopiramate-associated cognitive impairment. Hence, it is to be expectedthat the rate of cognitive impairment reported by this triplecombination would be less than that for topiramate on its own.

Ondansetron is disclosed herein as a drug useful alone or as part ofcombination drug therapy. Ondansetron is a 5-HT₃ receptor antagonist andhas functionally opposite effects to SSRIs and blocks serotonin agonismat the 5-HT₃ receptor. The dosage and treatment regimen foradministering ondansetron when it is being used as one compound of acombination therapy can be varied based on the other drug or drugs withwhich it is being administered, or based on other criteria such as theage, sex, health, and weight of the subject. The present inventiontherefore provides for the use of ondansetron at varying doses such asabout 0.01 μg/kg, about 0.1 μg/kg, about 1.0 μg/kg, about 5.0 μg/kg,about 10.0 μg/kg, about 0.1 mg/kg, about 1.0 mg/kg, about 5.0 mg/kg, andabout 10.0 mg/kg. In another embodiment, ondansetron is administered ata dosage ranging from about 0.01 μg/kg to about 100 μg/kg perapplication. In one aspect, ondansetron is administered at a dosageranging from about 0.1 μg/kg to about 10.0 μg/kg per application. In yetanother aspect, ondansetron is administered at a dosage ranging fromabout 1.0 μg/kg to about 5.0 μg/kg per application. In a further aspect,ondansetron is administered at a dosage of about 4.0 μg/kg perapplication. In another aspect, ondansetron is administered at a dosageof about 3.0 μg/kg per application. In one aspect, ondansetron isadministered at a dose of about 4 μg/kg twice daily (about 0.25 to 0.6mg twice daily for body weights between about 50 kg and 150 kg).

Ondansetron (C₁₈H₁₉N₃O; CAS Registry No. 99614-02-5; IUPAC name:9-methyl-3-[(2-methyl-1H-imidazol-1-yl)methyl]-1,2,3,9-tetrahydrocarbazol-4-one)has the following structure:

The present invention further provides for the use of other drugs suchas naltrexone as part of the drug combination therapy disclosed herein.In one embodiment, naltrexone is administered at a dose of about 10mg/day. In one aspect, naltrexone is administered at a dosage at adosage of about 50 mg/day. In one aspect, naltrexone is administered ata dosage of about 100 mg/day. In one aspect, naltrexone is administeredat a dosage ranging from about 1 mg to about 300 mg per application. Inanother aspect, naltrexone is administered at a dosage ranging fromabout 10 mg to about 50 mg per application. In a further aspect of theinvention, naltrexone is administered at a dosage of about 25 mg perapplication. In one embodiment, naltrexone is administered at least oncea month. In a further embodiment, naltrexone is administered once amonth. In one embodiment, naltrexone is administered at least once aweek. In another embodiment, naltrexone is administered at least once aday. In a further embodiment, naltrexone is administered at least twicea day. In one aspect, naltrexone is administered twice day.

Naltrexone (C₂₀H₂₃NO₄;17-(Cyclopropylmethyl)-4,5a-epoxy-3,14-dihydroxymorphinan-6-onehydrochloride; CAS Registry No. 16590-41-3) has the following structure:

Naltrexone also has important adverse events—nausea and vomiting—thatreduce compliance to it. Indeed, about 15% of individuals in alcoholtrials are unable to tolerate a naltrexone dose of 50 mg/day. This hasled to the development of depot formulations that release naltrexoneslowly to reduce the incidence of nausea and vomiting. Nevertheless,these depot formulation(s) appear to have similar compliance rates tothe oral form of the medication. Importantly, ondansetron reduces nauseaand decreases vomiting by slowing gut motility. Therefore, a combinationthat adds ondansetron to naltrexone will diminish the nausea andvomiting caused by naltrexone. This is an important therapeutic advancebecause many more people will be able to tolerate the treatment due toincreased compliance, and higher doses than the typically administerednaltrexone dose of 50 mg/day can be given to improve the therapeuticresponse.

In one embodiment, the alcohol-related disease or disorder being treatedincludes, but is not limited to, early-onset alcoholic, late-onsetalcoholic, alcohol-induced psychotic disorder with delusions, alcoholabuse, excessive drinking, heavy drinking, problem drinking, alcoholintoxication, alcohol withdrawal, alcohol intoxication delirium, alcoholwithdrawal delirium, alcohol-induced persisting dementia,alcohol-induced persisting amnestic disorder, alcohol dependence,alcohol-induced psychotic disorder with hallucinations, alcohol-inducedmood disorder, alcohol-induced or associated bipolar disorder,alcohol-induced or associated posttraumatic stress disorder,alcohol-induced anxiety disorder, alcohol-induced sexual dysfunction,alcohol-induced sleep disorder, alcohol-induced or associated gamblingdisorder, alcohol-induced or associated sexual disorder, alcohol-relateddisorder not otherwise specified, alcohol intoxication, and alcoholwithdrawal. In one aspect, the alcohol-related disease or disorder isearly onset alcoholic. In another aspect, the alcohol-related disease ordisorder is late onset alcoholic.

In one embodiment, the present invention provides compositions andmethods for reducing the frequency of alcohol consumption compared withthe frequency of alcohol consumption before the treatment. One ofordinary skill in the art will appreciate that the frequency can becompared with prior consumption by the subject or with consumption by acontrol subject not receiving the treatment. In one aspect, the type ofalcohol consumption is heavy drinking. In another aspect, it isexcessive drinking.

In one embodiment, the present invention provides compositions andmethods for reducing the quantity of alcohol consumed in a subjectcompared with the amount of alcohol consumed before the treatment orcompared with the alcohol consumption by a control subject not receivingthe treatment.

One of ordinary skill in the art will appreciate that in some instancesa subject being treated for and addictive disorder is not necessarilydependent. Such subjects include, for example, subjects who abusealcohol, drink heavily, drink excessively, are problem drinkers, or areheavy drug users. The present invention provides compositions andmethods for treating or preventing these behaviors in non-dependentsubjects.

In one embodiment of the invention, the present invention providescompositions and methods for improving the physical or psychologicalsequelae associated with alcohol consumption compared with a controlsubject not receiving the treatment.

In one embodiment, the present invention provides compositions andmethods for increasing the abstinence rate of a subject compared with acontrol subject not receiving the treatment.

In one embodiment, the present invention provides compositions andmethods for reducing the average level of alcohol consumption in asubject compared with the level of alcohol consumption before thetreatment or compared with the level of alcohol consumption by a controlsubject not receiving the treatment.

In one embodiment, the present invention provides compositions andmethods for reducing alcohol consumption and for increasing abstinencecompared with the alcohol consumption by the subject before treatment orwith a control subject not receiving the treatment.

In one embodiment, the present invention provides compositions andmethods for treating a subject with a predisposition to early-onsetalcoholism.

In one embodiment, the present invention provides compositions andmethods for treating a subject with a predisposition to late-onsetalcoholism.

One of ordinary skill in the art will appreciate that there are multipleparameters or characteristics of alcohol consumption which maycharacterize a subject afflicted with an alcohol-related disease ordisorder. It will also be appreciated that combination therapies may beeffective in treating more than one parameter, and that there aremultiple ways to analyze the effectiveness of treatment. The parametersanalyzed when measuring alcohol consumption or frequency of alcoholconsumption include, but are not limited to, heavy drinking days, numberof heavy drinking days, average drinking days, number of drinks per day,days of abstinence, number of individuals not drinking heavily orabstinent over a given time period, and craving. Both subjective andobjective measures can be used to analyze the effectiveness oftreatment. For example, a subject can self-report according toguidelines and procedures established for such reporting. The procedurescan be performed at various times before, during, and after treatment.Additionally, assays are available for measuring alcohol consumption.These assays include breath alcohol meter readings, measuring serum CDTand GGT levels, and measuring 5-HTOL urine levels.

In some embodiments, a first compound and a second compound areadministered nearly simultaneously. In other embodiments, a firstcompound is administered prior to the second compound. In yet otherembodiments, the first compound is administered subsequent to the secondcompound. If three or more compounds are administered, one of ordinaryskill in the art will appreciate that the three or more compounds can beadministered simultaneously or in varying order.

In certain embodiments disclosed herein, an individual is given apharmaceutical composition comprising a combination of two or morecompounds to treat or prevent an addiction-related disease or disorderor impulse control-related disease or disorder. In some of theseembodiments, each compound is a separate chemical entity. However, inother embodiments, the at least two compounds can be joined together bya chemical linkage, such as a covalent bond, so that the at least twodifferent compounds form separate parts of the same molecule. In oneaspect, the chemical linkage is selected such that after entry into thebody, the linkage is broken, such as by enzymatic action, acidhydrolysis, base hydrolysis, or the like, and the two separate compoundsare then formed.

Data from previous structure-activity relationship (SAR) studies withinthe art may be used as a guide to determine which compounds to use andthe optimal position or positions on the molecules to attach the tethersuch that potency and selectivity of the compounds will remain high. Thetether or linker moiety is chosen from among those of demonstratedutility for linking bioactive molecules together. Disclosed herein arerepresentative compounds that can be attached together in differentcombinations to form heterobivalent therapeutic molecules.

Examples of linkers reported in the scientific literature includemethylene (CH₂)_(n) linkers (Hussey et al., J. Am. Chem. Soc., 2003,125:3692-3693; Tamiz et al., J. Med. Chem., 2001, 44:1615-1622), oligoethyleneoxy O(—CH₂CH₂O—)_(n) units used to link naltrexamine to otheropioids, glycine oligomers of the formula—NH—(COCH₂NH)_(n)COCH₂CH₂CO—(NHCH₂CO)_(n)— used to link opioidantagonists and agonists together ((a) Portoghese et al., Life Sci.,1982, 31:1283-1286. (b) Portoghese et al., J. Med. Chem., 1986,29:1855-1861), hydrophilic diamines used to link opioid peptidestogether (Stepinski et al., Internat. J. of Peptide & Protein Res.,1991, 38:588-92), rigid double stranded DNA spacers (Paar et al., J.Immunol., 2002, 169:856-864) and the biodegradable linker poly(L-lacticacid) (Klok et al., Macromolecules, 2002, 35:746-759). The attachment ofthe tether to a compound can result in the compound achieving afavorable binding orientation. The linker itself may or may not bebiodegradable. The linker may take the form of a prodrug and be tunablefor optimal release kinetics of the linked drugs. The linker may beeither conformationally flexible throughout its entire length or else asegment of the tether may be designed to be conformationally restricted(Portoghese et al., J. Med. Chem., 1986, 29:1650-1653).

With respect to alcohol-related disorders, including but not limited toalcohol abuse and alcohol dependence, at least two compounds selectedfrom the group consisting of topiramate, ondansetron, and naltrexone,and analogs, derivatives, and modifications thereof, andpharmaceutically acceptable salts thereof, can be used to decreaseethanol consumption associated with such alcohol-related disorders. Inone aspect, topiramate and ondansetron are used. Accordingly, thepresent invention provides a method for treating or preventingalcohol-related disorders based on ethanol consumption, comprisingadministering to a subject in need of such treatment or prevention aneffective amount of at least two compounds selected from the groupconsisting of topiramate, ondansetron, and naltrexone, and analogs,derivatives, and modifications thereof or a pharmaceutically acceptablesalt thereof. In a further aspect, the combination pharmacotherapytreatment is used in conjunction with behavioral modification ortherapy.

Additional types of compounds can be administered to treat further theaddiction-related diseases and disorders or to treat other diseases anddisorders. The additional types of compounds include, but are notlimited to, adrenergics, adrenocortical steroids, adrenocorticalsuppressants, aldosterone antagonists, amino acids, analeptics,analgesics, anorectic compounds, anorexics, anti-anxiety agents,antidepressants, antihypertensives, anti-intiammnatories, antinauseants,antineutropenics, antiobsessional agents, antiparkinsonians,antipsychotics, appetite suppressants, blood glucose regulators,carbonic anhydrase inhibitors, cardiotonics, cardiovascular agents,choleretics, cholinergics, cholinergic agonists, cholinesterasedeactivators, cognition adjuvants, cognition enhancers, hormones, memoryadjuvants, mental performance enhancers, mood regulators, neuroleptics,neuroprotectives, psychotropics, relaxants, sedative-hypnotics,stimulants, thyroid hormones, thyroid inhibitors, thyromimetics,cerebral ischemia agents, vasoconstrictors, and vasodilators.

In one embodiment, the present invention provides methods andcompositions useful for decreasing mesocorticolimbic dopamine activity.

In one embodiment, the present invention provides methods andcompositions useful for regulating mesocorticolimbic dopamine activity.

In one embodiment, the present invention provides methods andcompositions useful for inhibiting glutamate function.

In one embodiment, the present invention provides methods andcompositions useful for facilitating γ-amino-butyric acid activity.

In one embodiment, the present invention provides methods andcompositions useful for regulating γ-amino-butyric acid activity.

The present invention provides for multiple methods for delivering thecompounds of the invention. The compounds may be provided, for example,as pharmaceutical compositions in multiple formats as well, including,but not limited to, tablets, capsules, pills, lozenges, syrups,ointments, creams, elixirs, suppositories, suspensions, inhalants,injections (including depot preparations), and liquids.

The present invention further encompasses biologically active analogs,homologs, derivatives, and modifications of the compounds of theinvention. Methods for the preparation of such compounds are known inthe art, in one aspect, the compounds are topiramate, naltrexone, andondansetron.

The compositions and methods described herein for treating or preventingalcohol-related diseases and disorders are also useful for treating orpreventing other addiction-related diseases and disorders and impulsecontrol disorders. In one aspect, the compositions and methods elicit anindirect effect on CMDA neurons. Such effects may be elicited, forexample, by regulating serotonergic, opiate, glutamate, orγ-amino-butyric acid receptors. In one aspect, the addictive diseasesand disorders include eating disorders, impulse control disorders,nicotine-related disorders, methamphetamine-related disordersamphetamine-related disorders, cannabis-related disorders,cocaine-related disorders, hallucinogen use disorders, inhalant-relateddisorders, benzodiazepine abuse or dependence related disorders, andopioid-related disorders.

A list of types of drugs, and specific drugs within categories which areencompassed within the invention is provided below.

Adrenergic: Adrenalone; Arnidephrine Mesylate; ApraclonidineHydrochloride; Brimonidine Tartrate; Dapiprazole Hydrochloride;Deterenol Hydrochloride; Dipivefrin; Dopamine Hydrochloride; EphedrineSulfate; Epinephrine; Epinephrine Bitartrate; Epinephryl Borate;Esproquin Hydrochloride; Etafedrine Hydrochloride; HydroxyamphetamineHydrobromide; Levonordefrin; Mephentermine Sulfate; MetaraminolBitartrate; Metizoline Hydrochloride; Naphazoline Hydrochloride;Norepinephrine Bitartrate; Oxidopamine; Oxymetazoline Hydrochloride;Phenylephrine Hydrochloride; Phenylpropanolamine Hydrochloride;Phenylpropanolamine Polistirex; Prenalterol Hydrochloride;Propylhexedrine; Pseudoephedrine Hydrochloride; TetrahydrozolineHydrochloride; Tramazoline Hydrochloride; Xylometazoline Hydrochloride.

Adrenocortical steroid: Ciprocinonide; Desoxycorticosterone Acetate;Desoxycorticosterone Pivalate; Dexamethasone Acetate; FludrocortisoneAcetate; Flumoxonide; Hydrocortisone Hemisuccinate; MethylprednisoloneHetnisuccinate; Naflocort; Procinonide; Timobesone Acetate; Tipredane.

Adrenocortical suppressant: Aminoglutethimide; Trilostane.

Alcohol deterrent: Disulfiram.

Aldosterone antagonist: Canrenoate Potassium; Canrenone; Dicirenone;Mexrenoate Potassium; Prorenoate Potassium; Spironolactone.

Amino acid: Alanine; Aspartic Acid; Cysteine Hydrochloride; Cystine;Histidine; Isoleucine; Leucine; Lysine; Lysine Acetate; LysineHydrochloride; Methionine; Phenylalanine; Proline; Serine; Threonine;Tryptophan; Tyrosine; Valine.

Analeptic: Modafinil.

Analgesic: Acetaminophen; Alfentanit Hydrochloride; AminobenzoatePotassium; Aminobenzoate Sodium; Anidoxime; Anileridine; AnileridineHydrochloride; Anilopam Hydrochloride; Anirolac; Antipyrine; Aspirin;Benoxaprofen; Benzydamine Hydrochloride; Bicifadine Hydrochloride;Brifentanil Hydrochloride; Bromadoline Maleate; Bromfenac Sodium;Buprenorphine Hydrochloride; Butacetin; Butixirate; Butorphanol;Butorphanol Tartrate; Carbamazepine; Carbaspirin Calcium; CarbipheneHydrochloride; Carfentanil Citrate; Ciprefadol Succinate; Ciramadol;Ciramadol Hydrochloride; Clonixeril; Clonixin; Codeine; CodeinePhosphate; Codeine Sulfate; Conorphone Hydrochloride; Cyclazocine;Dexoxadrol Hydrochloride; Dexpemedolac; Dezocine; Diflunisal;Dihydrocodeine Bitartrate; Dimefadane; Dipyrone; DoxpicomineHydrochloride; Drinidene; Enadoline Hydrochloride; Epirizole; ErgotamineTartrate; Ethoxazene Hydrochloride; Etofenamate; Eugenol; Fenoprofen;Fenoprofen Calcium; Fentanyl Citrate; Floctafenine; Flufenisal;Flunixin; Flunixin Meglumine; Flupirtine Maleate; Huproquazone;Huradoline Hydrochloride; Flurbiprofen; Hydromorphone Hydrochloride;Ibufenac; Indoprofen; Ketazocine; Ketorfanol; Ketorolac Tromethamine;Letimide Hydrochloride; Levomethadyl Acetate; Levomethadyl AcetateHydrochloride; Levonantradol Hydrochloride; Levorphanol Tartrate;Lofemizole Hydrochloride; Lofentanil Oxalate; Lorcinadol; Lomoxicam;Magnesium Salicylate; Mefenamic Acid; Menabitan Hydrochloride;Meperidine Hydrochloride; Meptazinol Hydrochloride; MethadoneHydrochloride; Methadyl Acetate; Methopholine; Methotrimeprazine;Metkephamid Acetate; Mimbane Hydrochloride; Mirfentanil Hydrochloride;Molinazone; Morphine Sulfate; Moxazocine; Nabitan Hydrochloride;Nalbuphine Hydrochloride; Nalmexone Hydrochloride; Namoxyrate; NantradolHydrochloride; Naproxen; Naproxen Sodium; Naproxol; NefopamHydrochloride; Nexeridine Hydrochloride; Noracymethadol Hydrochloride;Ocfentanil Hydrochloride; Octazamide; Olvanil; Oxetorone Fumarate;Oxycodone; Oxycodone Hydrochloride; Oxycodone Terephthalate; OxymorphoneHydrochloride; Pemedolac; Pentamorphone; Pentazocine; PentazocineHydrochloride; Pentazocine Lactate; Phenazopyridine Hydrochloride;Phenyramidol Hydrochloride; Picenadol Hydrochloride; Pinadoline;Pirfenidone; Piroxicam Olamine; Pravadoline Maleate; ProdilidineHydrochloride; Profadol Hydrochloride; Propirarn Fumarate; PropoxypheneHydrochloride; Propoxyphene Napsylate; Proxazole; Proxazole Citrate;Proxorphan Tartrate; Pyrroliphene Hydrochloride; RemifentanilHydrochloride; Salcolex; Salethamide Maleate; Salicylamide; SalicylateMeglumine; Salsalate; Sodium Salicylate; Spiradoline Mesylate;Sufentanil; Sufentanil Citrate; Talmetacin; Talniflumate; Talosalate;Tazadolene Succinate; Tebufelone; Tetrydamine; Tifurac Sodium; TilidineHydrochloride; Tiopinac; Tonazocine Mesylate; Tramadol Hydrochloride;Trefentanil Hydrochloride; Trolamine; Veradoline Hydrochloride;Verilopam Hydrochloride; Volazocine; Xorphanol Mesylate; XylazineHydrochloride; Zenazocine Mesylate; Zomepirac Sodium; Zucapsaicin.

Anorectic compounds including dexfenfluramine.

Anorexic; Aminorex; Amphecioral; Chlorphentermine Hydrochloride;Clominorex; Clortennine Hydrochloride; Diethylpropion Hydrochloride;Fenfluramine Hydrochloride; Fenisorex; Hudorex; Fluminorex;Levamfetamine Succinate; Mazindol; Mefenorex Hydrochloride;Phenmetrazine Hydrochloride; Phentermine; Sibutramine Hydrochloride.

Anti-anxiety agent: Adatanserin Hydrochloride; Alpidem; BinospironeMesylate; Bretazenil; Glemanserin; Ipsapirone Hydrochloride; MirisetronMaleate; Ocinapion; Ondansetron Hydrochloride; Panadiplon; Pancopride;Pazinaclone; Serazapine Hydrochloride; Tandospirone Citrate; ZalospironeHydrochloride.

Anti-cannabis agent: Rimonabant and other useful drugs, including drugsregulating the cannabinoid receptors.

Antidepressant: Adatanserin Hydrochloride; Adinazolam; AdinazolamMesylate; Alaproclate; Melamine Hydrochloride; Amedalin Hydrochloride;Amitriptyline Hydrochloride; Amoxapine; Aptazapine Maleate; AzaloxanFumarate; Azepindole; Azipramine Hydrochloride; BipenarnolHydrochloride; Bupropion Hydrochloride; Butacetin; ButriptylineHydrochloride; Caroxazone; Cartazolate; Ciclazindol; CidoxepinHydrochloride; Cilobamine Mesylate; Clodazon Hydrochloride;Clontipramine Hydrochloride; Cotinine Fumarate; Cyclindole; CypenamineHydrochloride; Cyprolidol Hydrochloride; Cyproximide; DaledalinTosylate; Dapoxetine Hydrochloride; Dazadrol Maleate; DazepinilHydrochloride; Desipramine Hydrochloride; Dexamisole; Deximafen;Dibenzepin Hydrochloride; Dioxadrol Hydrochloride; DothiepinHydrochloride; Doxepin Hydrochloride; Duloxetine Hydrochloride;Eclanamine Maleate; Encyprate; Etoperidone Hydrochloride; FantridoneHydrochloride; Fehmetozole Hydrochloride; Femmetramide; FezolamineFumarate; Fluotracen Hydrochloride; Fluoxetine; FluoxetineHydrochloride; Huparoxan Hydrochloride; Garafexine; Guanoxyfen Sulfate;Imafen Hydrochloride; Imiloxan Hydrochloride; Imipramine Hydrochloride;Indeloxazine Hydrochloride; Intriptyline Hydrochloride; Iprindole;Isocarboxazid; Ketipramine Fumarate; Lofepramine Hydrochloride;Lortalamine; Maprotiline; Maprotiline Hydrochloride; MelitracenHydrochloride; Milacemide Hydrochloride; Minaprine Hydrochloride;Mirtazapine; Moclobemide; Modaline Sulfate; Napactadine Hydrochloride;Napamezole Hydrochloride; Nefazodone Hydrochloride; Nisoxetine;Nitrafudam Hydrochloride; Nomifensine Maleate; NortriptylineHydrochloride; Octriptyline Phosphate; Opipramol Hydrochloride;Oxaprotiline Hydrochloride; Oxypertine; Paroxetine; Phenelzine Sulfate;Pirandamine Hydrochloride; Pizotyline; Pridefine Hydrochloride;Prolintane Hydrochloride; Protriptyline Hydrochloride; QuipazineMaleate; Rolicyprine; Seproxetine Hydrochloride; SertralineHydrochloride; Sibutramine Hydrochloride; Sulpiride; Suritozole;Tametraline Hydrochloride; Tampramine Fumarate; Tandamine Hydrochloride;Thiazesim Hydrochloride; Thozalinone; Tomoxetine Hydrochloride;Trazodone Hydrochloride; Trebenzomine Hydrochloride; Trimipramine;Trimipramine Maleate; Venlafaxine Hydrochloride; ViloxazineHydrochloride; Zimeldine Hydrochloride; Zometapine.

Antihypertensive: Aflyzosin Hydrochloride; Alipamide; Althiazide;Amiquinsin Hydrochloride; Amlodipine Besylate; Amlodipine Maleate;Anaritide Acetate; Atiprosin Maleate; Belfosdil; Bemitradine; BendacalolMesylate; Bendroflumethiazide; Benzthiazide; Betaxolol Hydrochloride;Bethanidine Sulfate; Bevantolol Hydrochloride; Biclodil Hydrochloride;Bisoprolol; Bisoprolol Fumarate; Bucindolol Hydrochloride; Bupicomide;Buthiazide; Candoxatril; Candoxatrilat; Captopril; Carvedilol;Ceronapril; Chlorothiazide Sodium; Cicletanine; Cilazapril; Clonidine;Clonidine Hydrochloride; Clopamide; Cyclopenthiazide; Cyclothiazide;Darodipine; Debrisoquin Sulfate; Delapril Hydrochloride; Diapamide;Diazoxide; Dilevalol Hydrochloride; Diltiazem Malate; Ditekiren;Doxazosin Mesylate; Ecadotril; Enalapril Maleate; Enalaprilat;Enalkiren; Endralazine Mesylate; Epithiazide; Eprosartan; EprosartanMesylate; Fenoldopam Mesylate; Flavodilol Maleate; Flordipine;Flosequinan; Fosinopril Sodium; Fosinoprilat; Guanabenz; GuanabenzAcetate; Guanacline Sulfate; Guanadrel Sulfate; Guancydine; GuanethidineMonosulfate; Guanethidine Sulfate; Guanfacine Hydrochloride; GuanisoquinSulfate; Guanoclor Sulfate; Guanoctine Hydrochloride; Guanoxabenz;Guanoxan Sulfate; Guanoxyfen Sulfate; Hydralazine Hydrochloride;Hydralazine Polistirex; Hydroflumethiazide; Indacrinone; Indapamide;Indolaprif Hydrochloride; Indoramin; Indoramin Hydrochloride; IndorenateHydrochloride; Lacidipine; Leniquinsin; Leveromakalim; Lisinopril;Lofexidine Hydrochloride; Losartan Potassium; Losulazine Hydrochloride;Mebutamate; Mecamylamine Hydrochloride; Medroxalol; MedroxalolHydrochloride; Methalthiazide; Methyclothiazide; Methyldopa;Methyldopate Hydrochloride; Metipranolol; Metolazone; MetoprololFumarate; Metoprolol Succinate; Metyrosine; Minoxidil; MonatepilMaleate; Muzolimine; Nebivolol; Nitrendipine; Ofornine; PargylineHydrochloride; Pazoxide; Pelanserin Hydrochloride; Perindopril Erbumine;Phenoxybenzamine Hydrochloride; Pinacidil; Pivopril; Polythiazide;Prazosin Hydrochloride; Primidolol; Priziditol Hydrochloride; QuinaprilHydrochloride; Quinaprilat; Quinazosin Hydrochloride; QuineloraneHydrochloride; Quinpirole Hydrochloride; Quinuclium Bromide; Ramipril;Rauwolfia Serpentina; Reserpine; Saprisartan Potassium; SarabasinAcetate; Sodium Nitroprusside; Sulfinalol Hydrochloride; Tasosartan;Teludipine Hydrochloride; Temocapril Hydrochloride; TerazosinHydrochloride; Terlakiren; Tiamenidine; Tiamenidine Hydrochloride;Tierynafen; Tinabinol; Tiodazosin; Tipentosin Hydrochloride;Trichlormethiazide; Trimazosin Hydrochloride; Trimethaphan Camsylate;Trimoxamine Hydrochloride; Tripamide; Xipamide; Zankiren Hydrochloride;Zofenoprilat Arginine.

Anti-inflammatory: Alclofenac; Alclometasone Dipropionate; AlgestoneAcetonide; Alpha Amylase; Amcinafal; Amicinafide; Amfenac Sodium;Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone;Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride;Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone;Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac;Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort;Desonide; Desoximetasone; Dexamethasone Dipropionate; DiclofenacPotassium; Diclofenac Sodium; Diflorasone Diacetate; Difiumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; FluocortinButyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; HalobetasolPropionate; Halopredone Acetate; Ibufenac; Ibuprofen; IbuprofenAluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; IndomethacinSodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam;Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanaic; Momiflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodotic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac;Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap;Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac;Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; Zomepirac Sodium.

Antinauseant: Buclizine Hydrochloride; Cyclizine Lactate; NaboctateHydrochloride.

Antineutropenic: Filgrastim; Lenograstim; Molgramostim; Regramostim;Sargramostim.

Antiobsessional agent: Fluvoxamine Maleate.

Antiparkinsonian: Benztropine Mesylate; Biperiden; BiperidenHydrochloride; Biperiden Lactate; Carmantadine; Ciladopa Hydrochloride;Dopamantine; Ethopropazine Hydrochloride; Lazabemide; Levodopa;Lometraline Hydrochloride; Mofegiline Hydrochloride; NaxagolideHydrochloride; Pareptide Sulfate; Procyclidine Hydrochloride;Quinetorane Hydrochloride; Ropinirole Hydrochloride; SelegilineHydrochloride; Tolcapone; Trihexyphenidyl Hydrochloride.Antiperistaltic: Difenoximide Hydrochloride; Difenoxin; DiphenoxylateHydrochloride; Fluperamide; Lidamidine Hydrochloride; LoperamideHydrochloride; Malethamer; Nufenoxole; Paregoric.

Antipsychotic: Acetophenazine Maleate; Alentemol Hydrobromide;Alpertine; Azaperone; Batelapine Maleate; Benperidol; BenzindopyrineHydrochloride; Brofbxine; Bromperidol; Bromperidol Decanoate; ButaclamolHydrochloride; Butaperazine; Butaperazine Maleate; Carphenazine Maleate;Carvotroline Hydrochloride; Chlorpromazine; ChlorpromazineHydrochloride; Chlorprothixene; Cinperene; Cintriamide; ClomacranPhosphate; Clopenthixol; Clopimozide; Clopipazan Mesylate; CloroperoneHydrochloride; Clothiapine; Clothixamide Maleate; Clozapine;Cyclophenazine Hydrochloride; Droperidol; Etazolate Hydrochloride;Fenimide; Flucindole; Flumezapine; Fluphenazine Decanoate; FluphenazineEnanthate; Fluphenazine Hydrochloride; Fluspiperone; Fluspirilene;Flutroline; Gevotroline Hydrochloride; Halopemide; Haloperidol;Haloperidol Decanoate; Iloperidone; lmidoline Hydrochloride; Lenperone;Mazapertine Succinate; Mesoridazine; Mesoridazine Besylate; Metiapine;Milenperone; Milipertine; Molindone Hydrochloride; NaranolHydrochloride; Neflumozide Hydrochloride; Ocaperidone; Olanzapine;Oxiperomide; Penfluridol; Pentiapine Maleate; Perphenazine; Pimozide;Pinoxepin Hydrochloride; Pipamperone; Piperacetazine; PipotiazinePalniitate; Piquindone Hydrochloride; Prochlorperazine Edisylate;Prochlorperazine Maleate; Promazine Hydrochloride; Remoxipride;Remoxipride Hydrochloride; Rimcazole Hydrochloride; SeperidolHydrochloride; Sertindole; Setoperone; Spiperone; Thioridazine;Thioridazine Hydrochloride; Thiothixene; Thiothixene Hydrochloride;Tioperidone Hydrochloride; Tiospirone Hydrochloride; TrifluoperazineHydrochloride; Trifluperidol; Triflupromazine; TrifluprotnazineHydrochloride; Ziprasidone Hydrochloride.

Appetite suppressant: Dexfenfluramine Hydrochloride; PhendimetrazineTartrate; Phentermine Hydrochloride.

Blood glucose regulators: Human insulin; Glucagon; Tolazamide;Tolbutamide; Chloropropamide; Acetohexamide and Glipizide.

Carbonic anhydrase inhibitor: Acetazolamide; Acetazolamide Sodium,Dichlorphenamide; Dorzolamide Hydrochloride; Methazolamide; SezolarmideHydrochloride.

Cardiac depressant: Acecainide Hydrochloride; Acetylcholine Chloride;Actisomide; Adenosine; Amiodarone; Aprindine; Aprindine Hydrochloride;Artilide Fumarate; Azimilide Dihydrochloride; Bidisomide; BucainideMaleate; Bucromarone; Butoprozine Hydrochloride; Capobenate Sodium;Capobenic Acid; Cifenline; Cifenline Succinate; Clofilium Phosphate;Disobutamide; Disopyramide; Disopyramide Phosphate; Dofetilide;Drobuline; Edifolone Acetate; Emilium Tosylate; Encainide Hydrochloride;Flecainide Acetate; Ibutilide Fumarate; Indecainide Hydrochloride;Ipazilide Fumarate; Lorajmine Hydrochloride; Lorcainide Hydrochloride;Meobentine Sulfate; Mexiletine Hydrochloride; Modecainide; Moricizine;Oxiramide; Pirmenol Hydrochloride; Pirolazamide; Pranolium Chloride;Procainamide Hydrochloride; Propafenone Hydrochloride; Pyrinoline;Quindonium Bromide; Quinidine Gluconate; Quinidine Sulfate; RecainamHydrochloride; Recainam Tosylate; Risotilide Hydrochloride; RopitoinHydrochloride; Sematilide Hydrochloride; Suricainide Maleate; Tocainide;Tocainide Hydrochloride; Transcainide.

Cardiotonic: Actodigin; Amrinone; Bemoradan; Butopamine; Carbazeran;Carsatrin Succinate; Deslanoside; Digitalis; Digitoxin; Digoxin;Dobutamine; Dobutamine Hydrochloride; Dobutamine Lactobionate;Dobutamine Tartrate; Enoximone; Imazodan Hydrochloride; lndolidan;Isomazole Hydrochloride; Levdobutamine Lactobionate; Lixazinone Sulfate;Medorinone; Milrinone; Pelrinone Hydrochloride; Pimobendan; Piroximone;Prinoxodan; Proscillaridin; Quazinone; Tazolol Hydrochloride;Vesnarinone.

Cardiovascular agent: Dopexamine; Dopexamine Hydrochloride.

Choleretic: Dehydrocholic Acid; Fencibutirol; Hymecromone; Piprozolin;Sincalide; Tocamphyl.

Cholinergic: Aceclidine; Bethanechol Chloride; Carbachol; DemecariumBromide; Dexpanthenol; Echothiophate Iodide; Isoflurophate; MethacholineChloride; Neostigmine Bromide; Neostigmine Methylsulfate; Physostigmine;Physostigmine Salicylate; Physostigmine Sulfite; Pilocarpine;Pilocarpine Hydrochloride; Pilocarpine Nitrate; Pyridostigmine Bromide.

Cholinergic agonist: Xanomeline; Xanomeline Tartrate.

Cholinesterase Deactivator: Obidoxime Chloride; Pralidoxime Chloride;Pralidoxime Iodide; Pralidoxime Mesylate.

Coccidiostat: Arprinocid; Narasin; Semduramicin; Semduramicin Sodium.

Cognition adjuvant: Ergoloid Mesylates; Piracetam; PramiracetamHydrochloride; Pramiracetam Sulfate; Tacrine Hydrochloride.

Cognition enhancer: Besipirdine Hydrochloride; Linopirdine; Sibopirdine.

Dopamine receptor agonist: cabergoline (Dostinex)

Hormone: Diethylstilbestrol; Progesterone; 17-hydroxy progesterone;Medroxyprogesterone; Norgestrel; Norethynodrel; Estradiol; Megestrol(Megace); Norethindrone; Levonorgestrel; Ethyndiol; Ethinyl estradiol;Mestranol; Estrone; Equilin; 17-alpha-dihydroequilin; equilenin;17-alpha-dihydroequilenin; 17-alpha-estradiol; 17-beta-estradiol;Leuprolide (lupron); Glucagon; Testolactone; Clomiphene; Han menopausalgonadotropins; Human chorionic gonadotropin; Urofollitropin;Bromocriptine; Gonadorelin; Luteinizing hormone releasing hormone andanalogs; Gonadotropins; Danazol; Testosterone; Dehydroepiandrosterone;Androstenedione; Dihydroestosterone; Relaxin; Oxytocin; Vasopressin;Folliculostatin; Follicle regulatory protein; Gonadoctrinins; Oocytematuration inhibitor; Insulin growth factor; Follicle StimulatingHormone; Luteinizing hormone; Tamoxifen; Corticorelin Ovine Triftutate;Cosyntropin; Metogest; Pituitary; Posterior; Seractide Acetate;Somalapor; Somatrem; Somatropin; Somenopor; Somidobove.

Memory adjuvant: Dimoxamine Hydrochloride; Ribaminol.

Mental performance enhancer: Aniracetam.

Mood regulator: Fengabine.

Neuroleptic: Duoperone Fumarate; Risperidone.

Neuroprotective: Dizocilpine Maleate.

Psychotropic: Minaprine.

Relaxant: Adiphenine Hydrochloride; Alcuronium Chloride; Aminophylline;Azumolene Sodium; Baclofen; Benzoctamine Hydrochloride; Carisoprodol;Chlorphenesin Carbamate; Chlorzoxazone; Cinflumide; Cinnamedrine;Clodanolene; Cyclobenzaprine Hydrochloride; Dantrolene; DantroleneSodium; Fenalanide; Fenyripol Hydrochloride; Fetoxylate Hydrochloride;Flavoxate Hydrochloride; Fletazepam; Flumetramide; —FlurazepamHydrochloride; Hexafluorenium Bromide; Isomylamine Hydrochloride;Lorbamate; Mebeverine Hydrochloride; Mesuprine Hydrochloride;Metaxalone; Methocarbamol; Methixene Hydrochloride; Nafomine Malate;Nelezaprine Maleate; Papaverine Hydrochloride; Pipoxolan Hydrochloride;Quinctolate; Ritodrine; Ritodrine Hydrochloride; Rolodine; TheophyllineSodium Glycinate; Thiphenamil Hydrochloride; Xitobam.

Sedative-hypnotic: Allobarbital; Alonimid; Alprazolam; AmobarbitalSodium; Bentazepam; Brotizolam; Butabarbital; Butabarbital Sodium;Butalbital; Capuride; Carbocloral; Chloral Betaine; Chloral Hydrate;Chlordiazepoxide Hydrochloride; Cloperidone Hydrochloride; Clorethate;Cyprazepam; Dexclamol Hydrochloride; Diazepam; Dichloralphenazone;Estazolam; Ethchlorvynol; Etomidate; Fenoham; Flunitrazepam; Fosazepam;Glutethimide; Halazepam; Lormetazepam; Mecloqualone; Meprobamate;Methaqualone; Midaflur; Paraldehyde; Pentobarbital; PentobarbitalSodium; Perlapine; Prazepam; Quazepam; Reclazepam; Roletamide;Secobarbital; Secobarbital Sodium; Suproclone; Thalidomide; Tracazolate;Trepipam Maleate; Triazolam; Tricetamide; Triclofos Sodium; Trimetozine;Uldazepam; Zaleplon; Zolazepam Hydrochloride; Zolpidem Tartrate.

Serotonin antagonist: Altanserin Tartrate; Amesergide; Ketanserin;Ritanserin.

Serotonin inhibitor: Cinanserin Hydrochloride; Fenclonine; FonazineMesylate; Xylamidine Tosylate.

Serotonin receptor antagonist: Tropanserin Hydrochloride.

Stimulant: Amfonelic Acid; Amphetamine Sulfate; Ampyzine Sulfate;Arbutamine Hydrochloride; Azabon; Caffeine; Ceruletide; CeruletideDiethylamine; Cisapride; Dazopride Fumarate; Dextroamphetamine;Dextroamphetamine Sulfate; Difluanine Hydrochloride; DimeflineHydrochloride; Doxapram Hydrochloride; Etryptamine Acetate; Ethamivan;Fenethylline Hydrochloride; Flubanilate Hydrochloride; Flurothyl;Histamine Phosphate; Indriline Hydrochloride; Mefexamide;Methamphetamine Hydrochloride; Methylphenidate Hydrochloride; Pemoline;Pyrovalerone Hydrochloride; Xamoterol; Xamoterol Fumarate. Synergist:Proadifen Hydrochloride.

Thyroid hormone: Levothyroxine Sodium; Liothyronine Sodium; Liotrix.

Thyroid inhibitor: Methimazole; Propyithiouracil.

Thyromimetic: Thyromedan Hydrochloride.

Cerebral ischemic agents: Dextrorphan Hydrochloride.

Vasoconstrictor: Angiotensin Amide; Felypressin; Methysergide;Methysergide Maleate.

Vasodilator: Alprostadil; Azaclorzine Hydrochloride; Bamethan Sulfate;Bepridil Hydrochloride; Buterizine; Cetiedil Citrate; ChromonarHydrochloride; Clonitrate; Diltiazem Hydrochloride; Dipyridamole;Droprenilamine; Erythrityl Tretranitrate; Felodipine; FlunarizineHydrochloride; Fostedil; Hexobendine; Inositol Niacinate; IproxamineHydrochloride; Isosorbide Dinitrate; Isosorbide Mononitrate; IsoxsuprineHydrochloride; Lidoflazine; Mefenidil; Mefenidil Fumarate; MibefradilDihydrochloride; Mioflazine Hydrochloride; Mixidine; Nafronyl Oxalate;Nicardipine Hydrochloride; Nicergoline; Nicorandil; Nicotinyl Alcohol;Nifedipine; Nimodipine; Nisoldipine; Oxfenicine; OxprenololHydrochloride; Pentaerythritol Tetranitrate; Pentoxifylline;Pentrinitrol; Perhexiline Maleate; Pindolol; Pirsidomine; Prenylamine;Propatyl Nitrate; Suloctidil; Terodiline Hydrochloride; TipropidilHydrochloride; Tolazoline Hydrochloride; Xanthinol Niacinate.

Assays and methods for testing compounds of the invention are describedherein or are known in the art. For example, see Lippa et al., U.S. Pat.Pub. No. 2006/0173-64, published Aug. 3, 2006.

The invention further encompasses treating and preventing obesity, i.e.,for affecting weight loss and preventing weight gain. Obesity is adisorder characterized by the accumulation of excess fat in the body.Obesity has been recognized as one of the leading causes of disease andis emerging as a global problem. Increased instances of complicationssuch as hypertension, non insulin-dependent diabetes mellitus,arteriosclerosis, dyslipidemia, certain forms of cancer, sleep apnea,and osteoarthritis have been related to increased instances of obesityin the general population. In one aspect, the invention encompassesadministering to a subject in need thereof a combination therapy toinduce weight loss. For example, subjects having a BMI of greater thanabout 25 (25.0-29.9 is considered overweight) are identified fortreatment. In one aspect, the individuals have a BMI of greater than 30(30 and above is considered obese). In another aspect, a subject may betargeted for treatment to prevent weight gain. In one embodiment, anindividual is instructed to take at least one compound of the inventionat least once daily and at least a second compound of the invention atleast once daily. The compound may be in the form of, for example, atablet, a lozenge, a liquid, etc. In one aspect, a third compound isalso taken daily. In one embodiment, compounds may be taken more thanonce daily. In another embodiment, compounds are taken less than oncedaily. The dosages can be determined based on what is known in the artor what is determined to be best for a subject of that age, sex, health,weight, etc. Compounds useful for treating obesity according to themethods of the invention, include, but are not limited to, topiramate,naltrexone, and ondansetron. See Weber (U.S. Pat. Pub. No. 20070275970)and McElroy (U.S. Pat. No. 6,323,236) for additional information andtechniques for administering drugs useful for treating obesity,addictive disorders, and impulse control disorders, and for determiningdosage schemes.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl) amines, tri(substituted alkenyl) amines,cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,substituted cycloalkyl amines, disubstituted cycloalkyl amines,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amines, trisubstituted cycloalkenyl amines,aryl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group. Examples of suitable amines include, by way of exampleonly, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl)amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol,tromethamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-alkylglucamines, theobromine, purines, piperazine, piperidine,morpholine, N-ethylpiperidine, and the like. It should also beunderstood that other carboxylic acid derivatives would be useful in thepractice of this invention, for example, carboxylic acid amides,including carboxamides, lower alkyl carboxamides, dialkyl carboxamides,and the like.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

Psychosocial Intervention and Management

The drug combination treatments of the present invention can be furthersupplemented by providing to subjects a form of psychosocialintervention and/or management, such as Brief Behavioral ComplianceEnhancement Treatment (BBCET). BBCET, a standardized, manual-guided,brief (i.e., delivered in about 15 minutes), psychosocial adherenceenhancement procedure, emphasizes that medication compliance is crucialto changing participants' drinking behavior (Johnson et al., BriefBehavioral Compliance Enhancement Treatment (BBCET) manual. In: JohnsonB A, Ruiz P, Galanter M, eds. Handbook of clinical alcoholism treatment.Baltimore, Md.: Lippincott Williams & Wilkins; 2003, 282-301). Briefinterventions (Edwards et al., J. Stud. Alcohol. 1977, 38:1004-103:1)such as BBCET, have been shown to benefit treatment of alcoholdependence. BBCET was modeled on the clinical management condition inthe National Institute of Mental Health collaborative depression trial,which was used as an adjunct to the medication condition for that study(Fawcett et al. Psychopharmacol Bull. 1987, 23:309-324). BBCET has beenused successfully as the psychosocial treatment platform in thesingle-site and multi-site efficacy trials of topiramate for treatingalcohol dependence (Johnson et al., Lancet. 2003, 361:1677-1685; Johnsonet al., JAMA, 2007, 298:1641-1651). It is delivered by trainedclinicians, including nurse practitioners and other non-specialists.Uniformity and consistency of BBCET delivery are ensured by ongoingtraining and supervision. BBCET is copyrighted material (Johnson et al.,Brief Behavioral Compliance Enhancement Treatment (BBCET) manual. In:Johnson B A, Ruiz P, Galanter M, eds. Handbook of clinical alcoholismtreatment. Baltimore, Md.: Lippincott Williams & Wilkins; 2003,282-301).

The present invention further encompasses the use of psychosocialmanagement regimens other than BBCET, including, but not limited to,Cognitive Behavioral Coping Skills Therapy (CBT) (Project MATCH ResearchGroup. Matching Alcoholism Treatments to Client Heterogeneity: ProjectMATCH posttreatment drinking outcomes. J Stud Alcohol. 1997; 58:7-29),Motivational Enhancement Therapy (MET) (Project MATCH Research Group.Matching Alcoholism Treatments to Client Heterogeneity: Project MATCHposttreatment drinking outcomes. J. Stud. Alcohol. 1997, 58:7-29),Twelve-Step Facilitation Therapy (TSF) (Project MATCH Research Group.Matching Alcoholism Treatments to Client Heterogeneity: Project MATCHposttreatment drinking outcomes. J. Stud. Alcohol. 1997, 58:7-29),Combined Behavioral Intervention (CBI), (Anton et al., JAMA, 2006,295:2003-2017) Medical Management (MM) (Anton et al., JAMA, 2006,295:2003-2017), or the Biopsychosocial, Report, Empathy, Needs, Directadvice, and Assessment (BRENDA) model (Garbutt et al., JAMA, 2005,293:1617-1625). The present invention further encompasses the use ofalternative interventions such as hypnosis or acupuncture to assist intreating an addictive disease or disorder.

The psychosocial management programs can be used before, during, andafter treating the subject with the combination drug therapy of theinvention.

One of ordinary skill in the art will recognize that psychosocialmanagement procedures, as well as alternative interventions such ashypnosis or acupuncture, can also be used in conjunction withcombination drug therapy to treat addictive and impulse-relateddisorders other than alcohol-related diseases and disorders.

The present invention further encompasses the use of combinationpharmacotherapy and behavioral (psychosocial) intervention or trainingto treat other addictive and/or impulse control disorders.

For example, binge eating disorder (BED) is characterized by discreteperiods of binge eating during which large amounts of food are consumedin a discrete period of time and a sense of control over eating isabsent. Persons with bulimia nervosa have been reported to haveelectroencephalographic abnormalities and to display reduced bingeeating in response to the anti-epileptic drug phenytoin. In addition, incontrolled trials in patients with epilepsy, topiramate was associatedwith suppression of appetite and weight loss unrelated to binge eating.Ondansetron has been shown to reduce binge eating.

BED is a subset of a larger classification of mental disorders broadlydefined as Impulse Control Disorders (ICDs) characterized by harmfulbehaviors performed in response to irresistible impulses. It has beensuggested that ICDs may be related to obsessive-compulsive disorder orsimilarly, maybe forms of obsessive-compulsive disorders. It has alsobeen hypothesized that ICDs may be related to mood disorder or may beforms of affective spectrum disorder, a hypothesized family of disorderssharing at least one common physiologic abnormality with majordepression. In the Diagnostic and Statistical Manual of Mental Disorders(DSM-IV), the essential feature of an ICD is the failure to resist animpulse, drive, or temptation to perform an act that is harmful to theperson or to others. For most ICDs, the individual feels an increasingsense of tension or arousal before committing the act, and thenexperiences pleasure, gratification, or release at the time ofcommitting the act. After the act is performed, there may or may not beregret or guilt. ICDs are listed in a residual category, the ICDs NotElsewhere Classified, which includes intermittent explosive disorder(IED), kleptomania, pathological gambling, pyromania, trichotillomania,and ICDs not otherwise specified (NOS). Examples of ICDs NOS arecompulsive buying or shopping, repetitive self-mutilation, nonparaphilicsexual addictions, severe nail biting, compulsive skin picking,personality disorders with impulsive features, attentiondeficit/hyperactivity disorder, eating disorders characterized by bingeeating, and substance use disorders.

Many drugs can cause physical and/or psychological addiction. Those mostwell known drugs include opiates, such as heroin, opium and morphine;sympathomimetics, including cocaine and amphetamines;sedative-hypnotics, including alcohol, benzodiazepines, andbarbiturates; and nicotine, which has effects similar to opioids andsympathomimetics. Drug addiction is characterized by a craving orcompulsion for taking the drug and an inability to limit its intake.Additionally, drug dependence is associated with drug tolerance, theloss of effect of the drug following repeated administration, andwithdrawal, the appearance of physical and behavioral symptoms when thedrug is not consumed. Sensitization occurs if repeated administration ofa drug leads to an increased response to each dose. Tolerance,sensitization, and withdrawal are phenomena evidencing a change in thecentral nervous system resulting from continued use of the drug. Thischange motivates the addicted individual to continue consuming the drugdespite serious social, legal, physical, and/or professionalconsequences.

Attention-deficit disorders include, but are not limited to,Attention-Deficit/Hyperactivity Disorder, Predominately InattentiveType; Attention-Deficit/Hyperactivity Disorder, PredominatelyHyperactivity-Impulsive Type; Attention-Deficit/Hyperactivity Disorder,Combined Type; Attention-Deficit/Hyperactivity Disorder not otherwisespecified (NOS); Conduct Disorder; Oppositional Defiant Disorder; andDisruptive Behavior Disorder not otherwise specified (NOS).

Depressive disorders include, but are not limited to, Major DepressiveDisorder, Recurrent; Dysthymic Disorder; Depressive Disorder nototherwise specified (NOS); and Major Depressive Disorder, SingleEpisode.

Parkinson's disease includes, but is not limited to, neuroleptic-inducedparkinsonism.

Addictive disorders include, but are not limited to, eating disorders,impulse control disorders, alcohol-related disorders, nicotine-relateddisorders, amphetamine-related disorders, cannabis-related disorders,cocaine-related disorders, gambling, sexual disorders, hallucinogen usedisorders, inhalant-related disorders, and opioid-related disorders, allof which are further subclassified as listed below.

Eating disorders include, but are not limited to, Bulimia Nervosa,Nonpurging Type; Bulimia Nervosa, Purging Type; and Eating Disorder nototherwise specified (NOS).

Impulse control disorders include, but are not limited to, intermittentExplosive Disorder, Kleptomania, Pyromania, Pathological Gambling,Trichotillomania, and Impulse Control Disorder not otherwise specified(NOS).

Nicotine-related disorders include, but are not limited to, NicotineDependence, Nicotine Withdrawal, and Nicotine-Related Disorder nototherwise specified (NOS).

Amphetamine-related disorders include, but are not limited to,Amphetamine Dependence, Amphetamine Abuse, Amphetamine Intoxication,Amphetamine Withdrawal, Amphetamine Intoxication Delirium,Amphetamine-Induced Psychotic Disorder with delusions,Amphetamine-Induced Psychotic Disorders with hallucinations,Amphetamine-Induced Mood Disorder, Amphetamine-Induced Anxiety Disorder,Amphetamine-Induced Sexual Dysfunction, Amphetamine-Induced SleepDisorder, Amphetamine Related Disorder not otherwise specified (NOS),Amphetamine Intoxication, and Amphetamine Withdrawal.

Cannabis-related disorders include, but are not limited to, CannabisDependence; Cannabis Abuse; Cannabis Intoxication; Cannabis IntoxicationDelirium; Cannabis-Induced Psychotic Disorder, with delusions;Cannabis-Induced Psychotic Disorder with hallucinations;Cannabis-Induced Anxiety Disorder; Cannabis-Related Disorder nototherwise specified (NOS); and Cannabis Intoxication.

Cocaine-related disorders include, but are not limited to, CocaineDependence, Cocaine Abuse, Cocaine Intoxication, Cocaine Withdrawal,Cocaine Intoxication Delirium, Cocaine-Induced Psychotic Disorder withdelusions, Cocaine-Induced Psychotic Disorders with hallucinations,Cocaine-Induced Mood Disorder, Cocaine-Induced Anxiety Disorder,Cocaine-Induced Sexual Dysfunction, Cocaine-Induced Sleep Disorder,Cocaine-Related Disorder not otherwise specified (NOS), CocaineIntoxication, and Cocaine Withdrawal.

Hallucinogen-use disorders include, but are not limited to, HallucinogenDependence, Hallucinogen Abuse, Hallucinogen Intoxication, HallucinogenWithdrawal, Hallucinogen Intoxication Delirium, Hallucinogen-InducedPsychotic Disorder with delusions, Hallucinogen-Induced PsychoticDisorder with hallucinations, Hallucinogen-Induced Mood Disorder,Hallucinogen-Induced Anxiety Disorder, Hallucinogen-Induced SexualDysfunction, Hallucinogen-Induced Sleep Disorder, Hallucinogen RelatedDisorder not otherwise specified (NOS), Hallucinogen Intoxication, andHallucinogen Persisting Perception Disorder (Flashbacks).

Inhalant-related disorders include, but are not limited to, InhalantDependence; Inhalant Abuse; Inhalant Intoxication; Inhalant IntoxicationDelirium, Inhalant-Induced Psychotic Disorder, with delusions;Inhalant-Induced Psychotic Disorder with hallucinations;Inhalant-Induced Anxiety Disorder; Inhalant-Related Disorder nototherwise specified (NOS); and Inhalant Intoxication.

Opioid-related disorders include, but are not limited to, OpioidDependence, Opioid Abuse, Opioid Intoxication, Opioid IntoxicationDelirium, Opioid-Induced Psychotic Disorder, with delusions,Opioid-Induced Psychotic Disorder with hallucinations, Opioid-InducedAnxiety Disorder, Opioid-Related Disorder not otherwise specified (NOS),Opioid Intoxication, and Opioid Withdrawal.

Tic disorders include, but are not limited to, Tourette's Disorder,Chronic Motor or Vocal Tic Disorder, Transient Tic Disorder, TicDisorder not otherwise specified (NOS), Stuttering, Autistic Disorder,and Somatization Disorder.

The present invention further encompasses the treatment of at least twoaddictive diseases or disorders or impulse control disorderssimultaneously. For example, the present invention provides for thesimultaneous treatment of alcohol related disorders and weight control(see Examples).

The present invention also encompasses the use of the compounds andcombination therapies of the invention in circumstances where mandatorytreatment may be applicable. For example, a court may require that asubject be treated or take part in a treatment program using compoundsor combination therapies of the invention as part of a mandated therapyrelated to alcohol abuse, excessive drinking, drug use, etc. Moreparticularly, the invention encompasses forensic uses where a courtwould require a subject who has been convicted of driving under theinfluence to be subjected to the methods of the invention as part of acondition of bail, probation, treatment, etc.

The invention also encompasses the use of pharmaceutical compositionscomprising compounds of the invention to practice the methods of theinvention, the compositions comprising at least one appropriate compoundand a pharmaceutically-acceptable carrier.

Other methods useful for the practice of the invention can be found, forexample, in U.S. Pat. Pub. No. 2006/0173064 (Lippa et al.), U.S. Pat.No. 6,323,236 (McElroy), U.S. Pat. Pub. No. 2007/0275970, PCTapplication PCT/US/2008/052628 (Johnson et al.) filed Jan. 31, 2008, andPCT application PCT/US/2007/088100 (Johnson and Tiouririne), filed Dec.19, 2007.

In one embodiment, a composition of the invention may comprise onecompound of the invention. In another embodiment, a composition of theinvention may comprise more than one compound of the invention. In oneembodiment, additional drugs or compounds useful for treating otherdisorders may be part of the composition. In one embodiment, acomposition comprising only one compound of the invention may beadministered at the same time as another composition comprising at leastone other compound of the invention. In one embodiment, the differentcompositions may be administered at different times from one another.When a composition of the invention comprises only one compound of theinvention, an additional composition comprising at least one additionalcompound must also be used.

The pharmaceutical compositions useful for practicing the invention maybe, for example, administered to deliver a dose of between 1 ng/kg/dayand 100 mg/kg/day.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered, for example, systemically in oral solidformulations, or as ophthalmic, suppository, aerosol, topical or othersimilar formulations. In addition to the appropriate compounds, suchpharmaceutical compositions may contain pharmaceutically-acceptablecarriers and other ingredients known to enhance and facilitate drugadministration. Other possible formulations, such as nanoparticles,liposomes, resealed erythrocytes, and immunologically based systems mayalso be used to administer an appropriate compound, or an analog,modification, or derivative thereof according to the methods of theinvention.

Compounds which are identified using any of the methods described hereinmay be formulated and administered to a subject for treatment of thediseases disclosed herein. One of ordinary skill in the art willrecognize that these methods will be useful for other diseases,disorders, and conditions as well.

A “prodrug” refers to an agent that is converted into the parent drug invivo. Prodrugs are often useful because, in some situations, they may beeasier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the parent drug, or may demonstrate increased palatability or beeasier to formulate. An example, without limitation, of a prodrug wouldbe a compound of the present invention which is administered as an ester(the “prodrug”) to facilitate transmittal across a cell membrane wherewater solubility is detrimental to mobility but which then ismetabolically hydrolyzed to the carboxylic acid, the active entity, onceinside the cell where water-solubility is beneficial. A further exampleof a prodrug might be a short peptide (polyaminoacid) bonded to an acidgroup where the peptide is metabolized to provide the active moiety.

The invention encompasses the preparation and use of pharmaceuticalcompositions comprising a compound useful for treatment of the diseasesdisclosed herein as an active ingredient. Such a pharmaceuticalcomposition may consist of the active ingredient alone, in a formsuitable for administration to a subject, or the pharmaceuticalcomposition may comprise the active ingredient and one or morepharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs,and birds including commercially relevant birds such as chickens, ducks,geese, and turkeys.

One type of administration encompassed by the methods of the inventionis parenteral administration, which includes, but is not limited to,administration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration is contemplated to include, but is not limited to,subcutaneous, intraperitoneal, intramuscular, and intrasternalinjection, and kidney dialytic infusion techniques

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,inhalation, buccal, ophthalmic, intrathecal or another route ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingactive ingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is a discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject, or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Lactulose can also be used as a freely erodible filler and is usefulwhen the compounds of the invention are prepared in capsule form.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, and hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.,polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl para hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

In one aspect, a preparation in the form of a syrup or elixir or foradministration in the form of drops may comprise active ingredientstogether with a sweetener, which is preferably calorie-free, and whichmay further include methylparaben or propylparaben as antiseptics, aflavoring and a suitable color.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of a dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil in water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentsincluding naturally occurring gums such as gum acacia or gum tragacanth,naturally occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e. about 20° C.) and which isliquid at the rectal temperature of the subject (i.e. about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for vaginal administration. Such acomposition may be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or gel or cream or a solution for vaginalirrigation.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e. such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made bycombining the active ingredient with a pharmaceutically acceptableliquid carrier. As is well known in the art, douche preparations may beadministered using, and may be packaged within, a delivery deviceadapted to the vaginal anatomy of the subject. Douche preparations mayfurther comprise various additional ingredients including, but notlimited to, antioxidants, antibiotics, antifungal agents, andpreservatives.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, and intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle (e.g., sterile pyrogen free water) prior toparenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil in water or water in oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and preferably from about 1 toabout 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally, thepropellant may constitute about 50% to about 99.9% (w/w) of thecomposition, and the active ingredient may constitute about 0.1% toabout 20% (w/w) of the composition. The propellant may further compriseadditional ingredients such as a liquid non-ionic or solid anionicsurfactant or a solid diluent (preferably having a particle size of thesame order as particles comprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to about 500 micrometers. Such a formulation isadministered in the manner in which snuff is taken, i.e., by rapidinhalation through the nasal passage from a container of the powder heldclose to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as about 0.1% (w/w) and as much as about100% (w/w) of the active ingredient, and may further comprise one ormore of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, comprise about0.1% to about 20% (w/w) active ingredient, the balance comprising anorally dissolvable or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powder oran aerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or atomized formulations, whendispersed, preferably have an average particle or droplet size in therange from about 0.1 to about 200 nanometers, and may further compriseone or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1% to 1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for intramucosal administration. Thepresent invention provides for intramucosal administration of compoundsto allow passage or absorption of the compounds across mucosa. Such typeof administration is useful for absorption orally (gingival, sublingual,buccal, etc.), rectally, vaginally, pulmonary, nasally, etc.

In some aspects, sublingual administration has an advantage for activeingredients which in some cases, when given orally, are subject to asubstantial first pass metabolism and enzymatic degradation through theliver, resulting in rapid metabotization and a loss of therapeuticactivity related to the activity of the liver enzymes that convert themolecule into inactive metabolites, or the activity of which isdecreased because of this bioconversion.

In some cases, a sublingual route of administration is capable ofproducing a rapid onset of action due to the considerable permeabilityand vascularization of the buccal mucosa. Moreover, sublingualadministration can also allow the administration of active ingredientswhich are not normally absorbed at the level of the stomach mucosa ordigestive mucosa after oral administration, or alternatively which arepartially or completely degraded in acidic medium after ingestion of,for example, a tablet.

Sublingual tablet preparation techniques known from the prior art areusually prepared by direct compression of a mixture of powderscomprising the active ingredient and excipients for compression, such asdiluents, binders, disintegrating agents and adjuvants. In analternative method of preparation, the active ingredient and thecompression excipients can be dry- or wet-granulated beforehand. In oneaspect, the active ingredient is distributed throughout the mass of thetablet. WO 00/16750 describes a tablet for sublingual use thatdisintegrates rapidly and comprises an ordered mixture in which theactive ingredient is in the form of microparticles which adhere to thesurface of water-soluble particles that are substantially greater insize, constituting a support for the active microparticles, thecomposition also comprising a mucoadhesive agent. WO 00/57858 describesa tablet for sublingual use, comprising an active ingredient combinedwith an effervescent system intended to promote absorption, and also apH-modifier.

The compounds of the invention can be prepared in a formulation orpharmaceutical composition appropriate for administration that allows orenhances absorption across mucosa. Mucosal absorption enhancers include,but are not limited to, a bile salt, fatty acid, surfactant, or alcohol.In specific embodiments, the permeation enhancer can be sodium cholate,sodium dodecyl sulphate, sodium deoxycholate, taurodeoxycholate, sodiumglycocholate, dimethylsulfoxide or ethanol. In a further embodiment, acompound of the invention can be formulated with a mucosal penetrationenhancer to facilitate delivery of the compound. The formulation canalso be prepared with pH, optimized for solubility, drug stability, andabsorption through mucosa such as nasal mucosa, oral mucosa, vaginalmucosa, respiratory, and intestinal mucosa.

To further enhance mucosal delivery of pharmaceutical agents within theinvention, formulations comprising the active agent may also contain ahydrophilic low molecular weight compound as a base or excipient. Suchhydrophilic low molecular weight compounds provide a passage mediumthrough which a water-soluble active agent, such as a physiologicallyactive peptide or protein, may diffuse through the base to the bodysurface where the active agent is absorbed. The hydrophilic lowmolecular weight compound optionally absorbs moisture from the mucosa orthe administration atmosphere and dissolves the water-soluble activepeptide. The molecular weight of the hydrophilic low molecular weightcompound is generally not more than 10000 and preferably not more than3000. Exemplary hydrophilic low molecular weight compounds includepolyol compounds, such as oligo-, di- and monosaccharides such assucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose,D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentibiose,glycerin, and polyethylene glycol. Other examples of hydrophilic lowmolecular weight compounds useful as carriers within the inventioninclude N-methylpyrrolidone, and alcohols (e.g., oligovinyl alcohol,ethanol, ethylene glycol, propylene glycol, etc.). These hydrophilic lowmolecular weight compounds can be used alone or in combination with oneanother or with other active or inactive components of the intranasalformulation.

When a controlled-release pharmaceutical preparation of the presentinvention further contains a hydrophilic base, many options areavailable for inclusion. Hydrophilic polymers such as a polyethyleneglycol and polyvinyl pyrrolidone, sugar alcohols such as D-sorbitol andxylitol, saccharides such as sucrose, maltose, lactulose, D-fructose,dextran, and glucose, surfactants such as polyoxyethylene-hydrogenatedcastor oil, polyoxyethylene polyoxypropylene glycol, and polyoxyethylenesorbitan higher fatty acid esters, salts such as sodium chloride andmagnesium chloride, organic acids such as citric acid and tartaric acid,amino acids such as glycine, beta-alanine, and lysine hydrochloride, andaminosaccharides such as meglumine are given as examples of thehydrophilic base. Polyethylene glycol, sucrose, and polyvinylpyrrolidone are preferred and polyethylene glycol are further preferred.One or a combination of two or more hydrophilic bases can be used in thepresent invention.

The present invention contemplates pulmonary, nasal, or oraladministration through an inhaler. In one embodiment, delivery from aninhaler can be a metered dose.

An inhaler is a device for patient self-administration of at least onecompound of the invention comprising a spray inhaler (e.g., a nasal,oral, or pulmonary spray inhaler) containing an aerosol sprayformulation of at least one compound of the invention and apharmaceutically acceptable dispersant. In one aspect, the device ismetered to disperse an amount of the aerosol formulation by forming aspray that contains a dose of at least one compound of the inventioneffective to treat a disease or disorder encompassed by the invention.The dispersant may be a surfactant, such as, but not limited to,polyoxyethylene fatty acid esters, polyoxyethylene fatty acid alcohols,and polyoxyethylene sorbitan fatty acid esters. Phospholipid-basedsurfactants also may be used.

In other embodiments, the aerosol formulation is provided as a drypowder aerosol formulation in which a compound of the invention ispresent as a finely divided powder. The dry powder formulation canfurther comprise a bulking agent, such as, but not limited to, lactose,sorbitol, sucrose, and mannitol.

In another specific embodiment, the aerosol formulation is a liquidaerosol formulation further comprising a pharmaceutically acceptablediluent, such as, but not limited to, sterile water, saline, bufferedsaline and dextrose solution.

In further embodiments, the aerosol formulation further comprises atleast one additional compound of the invention in a concentration suchthat the metered amount of the aerosol formulation dispersed by thedevice contains a dose of the additional compound in a metered amountthat is effective to ameliorate the symptoms of disease or disorderdisclosed herein when used in combination with at least a first orsecond compound of the invention.

Thus, the invention provides a self administration method for outpatienttreatment of an addiction related disease or disorder such as analcohol-related disease or disorder. Such administration may be used ina hospital, in a medical office, or outside a hospital or medical officeby non-medical personnel for self administration.

Compounds of the invention will be prepared in a formulation orpharmaceutical composition appropriate for nasal administration. In afurther embodiment, the compounds of the invention can be formulatedwith a mucosal penetration enhancer to facilitate delivery of the drug.The formulation can also be prepared with pH optimized for solubility,drug stability, absorption through nasal mucosa, and otherconsiderations.

Capsules, blisters, and cartridges for use in an inhaler or insufflatormay be formulated to contain a powder mix of the pharmaceuticalcompositions provided herein; a suitable powder base, such as lactose orstarch; and a performance modifier, such as 1-leucine, mannitol, ormagnesium stearate. The lactose may be anhydrous or in the form of themonohydrate. Other suitable excipients include dextran, glucose,maltose, sorbitol, xylitol, fructose, sucrose, and trehalose. Thepharmaceutical compositions provided herein for inhaled/intranasaladministration may further comprise a suitable flavor, such as mentholand levomenthol, or sweeteners, such as saccharin or saccharin sodium.

For administration by inhalation, the compounds for use according to themethods of the invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the drugs and asuitable powder base such as lactose or starch.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

Typically, dosages of the compounds of the invention which may beadministered to an animal, preferably a human, range in amount fromabout 1.0 μg to about 100 g per kilogram of body weight of the animal.The precise dosage administered will vary depending upon any number offactors, including but not limited to, the type of animal and type ofdisease state being treated, the age of the animal and the route ofadministration. Preferably, the dosage of the compound will vary fromabout 1 mg to about 10 g per kilogram of body weight of the animal. Morepreferably, the dosage will vary from about 10 mg to about 1 g perkilogram of body weight of the animal.

The compounds may be administered to a subject as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the animal, etc.

The invention also includes a kit comprising the compounds of theinvention and an instructional material that describes administration ofthe compounds. In another embodiment, this kit comprises a (preferablysterile) solvent suitable for dissolving or suspending the compositionof the invention prior to administering the compound to the mammal.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression that can be usedto communicate the usefulness of the compounds of the invention in thekit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of alleviating the diseases ordisorders. The instructional material of the kit of the invention may,for example, be affixed to a container that contains a compound of theinvention or be shipped together with a container that contains thecompounds. Alternatively, the instructional material may be shippedseparately from the container with the intention that the instructionalmaterial and the compound be used cooperatively by the recipient.

Relevant nucleic acid and amino sequences encompassed by the presentinvention include, but are not limited to:

SEQ ID NO: 1-Human serotonin transporter (SLC6A4), nucleic acid sequence (GenBank Accession No. NM_001045-2775 bp mRNA)-acagccagcgccgccgggtgcctcgagggcgcgaggccagcccgcctgcccagcccgggaccagcctccccgcgcagcctggcaggtctcctggaggcaaggcgaccttgcttgccctctcttgcagaataacaaggggcttagccacaggagttgctggcaagtggaaagaagaacaaatgagtcaatcccgacgtgtcaatcccgacgatagagagctcggaggtgatccacaaatccaagcacccagagatcaattgggatccttggcagatggacatcagtgtcatttactaaccagcaggatggagacgacgcccttgaattctcagaagcagctatcagcgtgtgaagatggagaagattgtcaggaaaacggagttctacagaaggttgttcccaccccaggggacaaagtggagtccgggcaaatatccaatgggtactcagcagttccaagtcctggtgcgggagatgacacacggcactctatcccagcgaccaccaccaccctagtggctgagcttcatcaaggggaacgggagacctggggcaagaaggtggatttccttctctcagtgattggctatgctgtggacctgggcaatgtctggcgcttcccctacatatgttaccagaatggagggggggcattcctcctcccctacaccatcatggccatttttgggggaatcccgctcttttacatggagctcgcactgggacagtaccaccgaaatggatgcatttcaatatggaggaaaatctgcccgattttcaaagggattggttatgccatctgcatcattgccttttacattgcttcctactacaacaccatcatggcctgggcgctatactacctcatctcctccttcacggaccagctgccctggaccagctgcaagaactcctggaacactggcaactgcaccaattacttctccgaggacaacatcacctggaccctccattccacgtcccctgctgaagaattttacacgcgccacgtcctgcagatccaccggtctaaggggctccaggacctggggggcatcagctggcagctggccctctgcatcatgctgatcttcactgttatctacttcagcatctggaaaggcgtcaagacctctggcaaggtggtgtgggtgacagccaccttcccttatatcatcctttctgtcctgctggtgaggggtgccaccctccctggagcctggaggggtgttctcttctacttgaaacccaattggcagaaactcctggagacaggggtgtggatagatgcagccgctcagatcttcttctctcttggtccgggctttggggtcctgctggcttttgctagctacaacaagttcaacaacaactgctaccaagatgccctggtgaccagcgtggtgaactgcatgacgagcttcgtttcgggatttgtcatcttcacagtgctcggttacatggctgagatgaggaatgaagatgtgtctgaggtggccaaagacgcaggtcccagcctcctcttcatcacgtatgcagaagcgatagccaacatgccagcgtccactttctttgccatcatcttctttctgatgttaatcacgctgggcttggacagcacgtttgcaggcttggagggggtgatcacggctgtgctggatgagttcccacacgtctgggccaagcgccgggagcggttcgtgctcgccgtggtcatcacctgcttctttggatccctggtcaccctgacttttggaggggcctacgtggtgaagctgctggaggagtatgccacggggcccgcagtgctcactgtcgcgctgatcgaagcagtcgctgtgtcttggttctatggcatcactcagttctgcagggacgtgaaggaaatgctcggcttcagcccggggtggttctggaggatctgctgggtggccatcagccctctgtttctcctgttcatcatttgcagttttctgatgagcccgccacaactacgacttttccaatataattatccttactggagtatcatcttgggttactgcataggaacctcatctttcatttgcatccccacatatatagcttatcggttgatcatcactccagggacatttaaagagcgtattattaaaagtattaccccagaaacaccaacagaaattccttgtggggacatccgcttgaatgctgtgtaacacactcaccgagaggaaaaaggcttctccacaacctcctcctccagttctgatgaggcacgcctgccttctcccctccaagtgaatgagtttccagctaagcctgatgatggaagggccttctccacagggacacagtctggtgcccagactcaaggcctccagccacttatttccatggattcccctggacatattcccatggtagactgtgacacagctgagctggcctattttggacgtgtgaggatgtggatggaggtgatgaaaaccaccctatcatcagttaggattaggtttagaatcaagtctgtgaaagtctcctgtatcatttcttggtatgatcattggtatctgatatctgtttgcttctaaaggtttcactgttcatgaatacgtaaactgcgtaggagagaacagggatgctatctcgctagccatatattttctgagtagcatatataattttattgctggaatctactagaaccttctaatccatgtgctgctgtggcatcaggaaaggaagatgtaagaagctaaaatgaaaaatagtgtgtccatgcaaaaaaaaaaaSEQ ID NO: 2-Human serotonin transporter (SLC6A4), amino acid sequence (GenBank Accession No. NP_001036; 630 residues)-mettplnsqkqlsacedgedcqengvlqkvvptpgdkvesgqisngysavpspgagddtrhsipattttlvaelhqgeretwgkkvdfllsvigyavdlgnvwrfpyicyqngggafllpytimaifggiplfymelalgqyhrngcisiwrkicpifkgigyaiciiafyiasyyntimawalyylissftdqlpwtscknswntgnctnyfsednitwtlhstspaeefytrhvlqihrskglqdlggiswqlalcimliftviyfsiwkgvktsgkvvwvtatfpyiilsvllvrgatlpgawrgvlfylkpnwqklletgvwidaaaqiffslgpgfgvllafasynkfnnncyqdalvtsvvncmtsfvsgfviftvlgymaemrnedvsevakdagpsllfityaeaianmpastffaiifflmlitlgldstfaglegvitavldefphvwakrrerfvlavvitcffgslvtltfggayvvklleeyatgpavltvalieavavswfygitqfcrdvkemlgfspgwfwricwvaisplfllfiicsflmsppqlrlfqynypywsiilgycigtssficiptyiayrliitpgtfkeriiksitpetpteipcgdirlnav SEQ ID NO: 3-SNP rs25531 forward primer-5′-TCCTCCGCTTTGGCGCCTCTTCC-3′ (forward)SEQ ID NO: 4-SNP rs25531 reverse primer- 5′-TGGGGGTTGCAGGGGAGATCCTG-3′(reverse) SEQ ID NO: 5-rs2891483 forward primer- GCAGAAGCGATAGCCAACATGSEQ ID NO: 6-rs2891483 reverse primer- CAAGCCCAGCGTGATTAACATCSEQ ID NO: 7-rs2891483 probe- CTTTCTTTGCC[C/A]TCATCT(represented by CTTTCTTTGCCNTCATCT in the sequence listing)SEQ ID NO: 8-First primer for amplifying the 5′-HTTLPR44 bp promoter region repeat polymorphism-5′-CGT TGC CGC TCT GAA TGC CAG-3′SEQ ID NO: 9-Second primer for amplifying the 5′-HTTLPR44 bp promoter region repeat polymorphism-5′-GGA TTC TGG TGC CAC CTA GAC GCC-3′SEQ ID NO: 10-SNP polymorphism site of SLC6A4 rs1042173-GCCATATATTTTCTGAGTAGCATATA[G/T]AATTTTATTGCTGGAATCTACTAGA-(represented by GCCATATATTTTCTGAGTAGCATATANAATTTTATTGCTGGAATCTACTAGA in the sequence listing)

Other methods and techniques useful for the practice of the inventionthat are not described are known in the art, for example, seeInternational application no. PCT/US2008/064232.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples, therefore, specifically point out embodiments of the presentinvention, and are not to be construed as limiting in any way theremainder of the disclosure.

EXAMPLES Example 1 Correlation of a Functional Polymorphism in the 3′UTRof the Serotonin Transporter Gene SLC6A4 and its Association withDrinking Activity

It was determined whether allelic variation at a single nucleotidepolymorphism (SNP) within a putative polyadenylation signal for acommonly used 3′ polyadenylation site G2651T SNP (National Center forBiotechnology Information reference ID # rs1042173) of SLC6A4, theserotonin transporter gene, is associated with differences in theseverity of drinking among treatment-seeking alcoholics. To determinethe functional significance of the G2651T/rs1042173 SNP, we examinedwhether allelic variation at this site was associated with quantifiablechanges in mRNA expression level and 5-HTT protein expression. The humanserotonin transporter gene (SLC6A4) is found at position 17q11.1-q12 ofchromosome 17. G2651T/rs1042173 is in exon 15 position 25,549,137.Additionally, the 5-HTTLPR is in the promoter at chromosome position˜25,588,500. The sequence of the 5′-flanking region of the genecorresponds to GenBank Accession No. X76753 (see Heils et al., J.Neurochem., 66:2621, 1996).

Materials and Methods

Subjects

A total of two hundred seventy-five alcohol-dependent subjects (78.5%male) aged between 18 and 66 years were used in this study, in which 198of them were included in our previous study (Johnson et al., 2008). Allsubjects were considered to be alcohol-dependent (see below for details)and enrolled as part of a pharmacotherapy trial for the treatment ofalcohol dependence at both the University of Texas Health Science Centerat San Antonio and at University of Virginia. Participants wererecruited by newspaper or radio advertisements, and written informedconsent—approved by review boards of all participating institutes, wasobtained from all participants.

Alcohol dependence was diagnosed using the Structured Clinical Interviewfor Diagnostic and Statistical Manual of Mental Disorders, 4th edition(American Psychiatric Association, 1994) Axis I Disorders, by a trainedpsychologist. All subjects had a score of ≥8 on the Alcohol UseDisorders Identification Test (AUDIT) (Babor et al., 1992) that screenedfor individuals with alcohol use and related problems: reported heavydrinking which was defined as drinking of ≥21 standard drinks/week forwomen and ≥30 standard drinks/week for men during the 90 days prior toenrollment. Absence of other substance use was confirmed by negativeurine toxicological screen for narcotics, amphetamines, or sedativehypnotics at enrollment. The Subjects who met the following criteriawere excluded from the study: current axis I psychiatric diagnoses otherthan alcohol or nicotine dependence; significant alcohol withdrawalsymptoms based on the revised clinical institute withdrawal assessmentfor alcohol scale (Sullivan et al., 1989) score>15]; clinicallysignificant physical abnormalities based on physical examination,electrocardiogram recording, hematological assessment, biochemistryincluding serum bilirubin concentration, and urinalysis; pregnant orlactating state; treatment for alcohol dependence ≤30 days prior toenrollment, and mandated incarceration or employment loss for notreceiving alcohol treatment.

Drinking Measurements

Self-reported drinking (measured in standard drinks) in the 90 daysprior to study enrollment was quantified using the timeline follow-backmethod. One standard drink was defined as 0.35 L of beer, 0.15 L ofwine, or 0.04 L of 80-proof liquor. The intensity of drinking wasassessed by measurement of the mean drinks per drinking day and meandrinks per day. Drinks per drinking day was defined as the total numberof drinks divided by the number of drinking days within the 90 days;drinks per day was defined as the total number of drinks divided by 90days.

DNA Extraction and Genotyping

Ten milliliters of blood was drawn from each subject at baseline toobtain white blood cells for the determination of 5-HTT genotypes. DNAwas extracted using a Gentra Puregene® kit (QIAGEN Inc., Valencia,Calif.). SNPs for association analyses were selected using the NationalCenter for. Biotechnology Information (NCBI) dbSNP database(http://www.ncbi.nlm.nih.gov/SNP/) based on their functional potentialand minor allele frequency (MAI) ≥0.05. The average SNP density is ˜7kb. Detailed information on SNP locations, chromosomal positions,allelic variants, MAF and primer/probe sequences is summarized inTable 1. Four of the five SNPs (rs6354, rs6355, rs28914832, andrs1042173) were genotyped with TaqMart® SNP genotyping assays (AppliedBiosystems, Foster City, Calif.). Polymerase chain reaction (PCR)conditions were 50° C. for 2 min, 95° C. for 10 min, 30 cycles of 95° C.for 25 s, and 60° C. for 1 min. Alleles of each SNP were determined withan ABI PRISM® 7900HT instrument (Applied Biosystems) and analyzed usingsequence detection system (SDS) software.

The DNA samples from the 77 subjects that were not included in ourprevious study were genotyped for 5′-HTTLPR L/S alleles as describedpreviously (Johnson et al., 2008).

Assays for SNP rs25531 were carried out as described by Wendland et al(2006). Each assay had a total assay volume of 20 μl, and the PCRconditions were 15 min at 95° C., 35 cycles of 94° C. for 30 s, 65.5° C.for 90 s, and 72° C. for 60 s, with a final extension step of 10 min at72° C. Afterwards, 10 μl of PCR product was double-digested with HpaIIand BccI (5 U each; New England Biolabs, Ipswich, Mass.) in a 20-μlreaction assay containing NEBuffer 1 and bovine serum albumin at 37° C.for 5 h. Finally, 10 μl of remaining PCR product and 20 μl ofrestriction enzyme assay solution were electrophoresed with 3.5%UltraPure™ agarose gel (Invitrogen™, Carlsbad, Calif.) for 1.5-2 h at100 V in Tris/Borate/ethylenediaminetetraacetic acid buffer andvisualized by ethidium bromide staining (Sigma-Aldrich, St Louis, Mo.).The uncut PCR product in the lanes loaded with restrictionenzyme-digested PCR products were detected as the “A” allele of rs25531,and the cut product at 402 bp were detected as the “G” allele ofrs25531.

Association Analyses with Drinking Intensity

Associations of individual SNPs with the intensity of drinking (i.e.,drinks per drinking day and drinks per day) were analyzed using theanalysis of variance test in SAS version 9.1 (SAS Institute Inc., Cary,N.C.). Three genetic models (additive, dominant, and recessive) weretested using gender and age as covariates. Pair-wise linkagedisequilibrium (LD) among all 6 polymorphisms was assessed using theHaploview program (Barrett et al., 2005). All associations found to besignificant were corrected for multiple testing according to Bonferronicorrection by dividing the significance level by the number ofpolymorphisms studied.

Cloning, Cell Culture, and Transfection

Allelic expression differences of the SNP (rs1042173) that showed asignificant association with drinking intensity were studied using an invitro system. The human 5-HTT containing the G allele of rs1042173 inpBluescript II KS (−) was a generous gift from Prof. Randy D. Blakely(Vanderbilt University School of Medicine, Nashville, Tenn.). This 5-HTTcDNA/Bluescript construct contained the coding region as well as both5′- and 3′-untranslated regions of the gene with a total length of 2508bp. The human 5-HTT construct was digested with HindIII/XbaI andsubcloned into pcDNA3.1(−) (Invitrogen™) pre-digested with HindIII/XbaIas described by Qian et al (Qian et al., 1997). To produce plasmid withthe T allele of rs1042173, a DNA plasmid carrying the G allele wasmutated using the GeneTailor™ site-directed mutagenesis system(Invitrogen™). Both constructs were DNA sequence verified.

HeLa cells were cultured in complete medium [Dulbecco's modified Eagle'smedium (HyClone, Logan, Utah), 10% GIBCO® fetal bovine serum(Invitrogen™), 100 U/ml penicillin, and 100 μg/ml streptomycin(Mediatech, Inc., Manassas, Va.)] in 6-well plates and maintained in ahumidified incubator at 37° C. and 5% CO2. After the cells reachedapproximately 80% confluence, they were transfected with one of the twoalleles (4 μg of plasmids per well)) in 6-well culture plates usingLipofectamine™ 2000 (Invitrogen™) according to the manufacturer'sguidelines. RNA and proteins were extracted from HeLa cells 24 h aftertransfection.

RNA Isolation, Reverse Transcription, and Quantitative Real-TimePolymerase Chain Reaction (qRT-PCR)

Total RNA was extracted from HeLa cells with TRIZOL® reagent(Invitrogen™). Potential DNA contaminations were removed by treating theRNA samples with RNase-free DNase I at 37° C. for 30 min. Each RNAsample was reverse transcribed in vitro using SuperScript® II RT(Invitrogen™) to obtain cDNA. These cDNA samples were transcribed withTaqMan® Gene Expression Assays (Applied Biosystems) specific for 5-HTTmRNA, and the resulting 5-HTT mRNA was quantified by the ABI PRISM®7900HT sequence detection system. TaqMan® primer/probe sets forglyceraldehyde-3-phosphate dehydrogenase (G3PDH) were used as aninternal control to normalize the expression of 5-HTT. For each qRT-PCRexperiment, four samples with the G allele, four samples with the Tallele, and four controls with the pcDNA3.1 (−) vector only were used incell cultures from transfections carried out on different days.

Western Blotting Analysis

Radioimmunoprecipitation assay buffer [Tris-HCl (pH 7.4), 1% NP-40, 150mM NaCl, 0.25% Na-deoxycholate, and 1 mM EDTA] was added to HeLa cellsafter washing the cells once with ice-cold phosphate-buffered saline.The protein concentration of the cell lysates was determined using theBio-Rad assay (Bio-Rad Laboratories, Hercules, Calif.). Fifteenmicrograms of samples were loaded onto 10% sodium dodecylsulfate-polyacrylamide gels (30% acrylamide) in Tris-glycine buffercontaining sodium dodecyl sulfate. The separated proteins were thenelectrophoretically transferred to nitrocellulose membranes(PerkinElmer, Waltham, Mass.) overnight at 25 mA. The membranes wereblocked for 1 h at room temperature with 2% non-fat dry milk diluted inTris-buffered saline with Tween® 20 (TBST) buffer and washed three timesfor 10 min each in TBST buffer; then they were incubated overnight withprimary antibody (1:200) at 4° C. [rabbit polyclonal immunoglobulin G(IgG) corresponding to the C-terminus of a sodium-dependent 5-HTT ofhuman origin (200 μg/ml stock solution) (Santa Cruz Biotechnology, Inc.,Santa Cruz, Calif.)]. Membranes then were washed three times for 10 mineach in TBST buffer and incubated with secondary antibodies (1:5,000)[anti-rabbit IgG (goat), horseradish peroxidase labeled (PerkinElmer)]for 1.5 h at room temperature. The hybridized membranes were washed withTBST buffer four times for 10 min each, and the immunoreactivity of theproteins was detected using Western Lightning® Chemiluminescence ReagentPlus (PerkinElmer) and exposure to X-ray film. Tubulin protein was usedas an internal control to control for discrepancies in the loading ofproteins in each lane. A monoclonal antibody (mouse monoclonal antibodyto α-tubulin) was used as the primary antibody (1:2,000), and ananti-mouse IgG was used as the secondary antibody in western blottingfor tubulin.

Densitometric and Statistical Analysis

Western blotting films were scanned on a UMAX scanner (Techville, Inc.,Dallas, Tex.) using Adobe Photoshop (v. 6.0; Adobe Systems Inc., SanJose, Calif.), and the optical densities of the G and T alleles andtubulin were measured using NIH Image software (v. 1.61). The opticaldensities of bands of the G and T alleles and of tubulin were quantifiedusing densitometry. The background (the area surrounding each band)optical density values were quantified the same way as for the proteinbands, and the values were subtracted from the measured optical densityvalues for the protein bands. The ratios of the optical density valuesof the G and T alleles to the optical density values of tubulin in thecorresponding samples were calculated to normalize the expression of theG and T alleles of the 5-HTT. Student's t-test was used to analyzeprotein data to determine the significance of expression differencesbetween the G and T alleles.

Results

Genotyping and LD Analysis

DNA samples from 275 alcohol-dependent subjects were genotyped in thisstudy. Of these subjects, 165 were Caucasians (43 females and 122 males)and 110 were Hispanics (16 females and 94 males). Genotypicdistributions of all 5 SNPs and 5-HTTLPR L/S alleles, conformed to theHardy-Weinberg equilibrium (Table 1). Further, the LD analyses usingHaploview revealed no haplotype blocks among the 5 SNPs and the5′-HTTLPR US polymorphism according to the criteria of Gabriel et al.(2002), in Caucasian, Hispanic or pooled populations, respectively (FIG.1).

Associations with Self-Reported Drinking Measures

To exclude potential variations caused by ethnic differences on drinkingintensity, subgroups of subjects based on ethnicity were analyzedseparately for all polymorphisms studied here. Among the polymorphismsanalyzed individually using SAS (version 9.1) program for associationswith drinking intensity, only SNP rs1042173 in the 3′ UTR of SLC6A4showed a significant association with intensity of drinking. Table 2shows demographic and drinking parameters of the cohort analyzed forrs1042173 association studies. No significant association was detectedfor other genetic polymorphisms with intensity of drinking in Caucasian,Hispanic or pooled populations (data not shown).

Among Caucasian subjects, mean drinks per drinking day differedsignificantly among TT, TG, and GG genotypes (F=5.625; p=0.004). UsingTukey's post-hoc multiple comparison test, the differences between TGheterozygotes and TT homozygotes were statistically significant(d=4.721; p=0.002); however, the differences between TG heterozygotesand GG homozygotes were not (d=2.175; p=0.20). When TT and TG werecombined and compared with GG using Student's t-test, the means did notdiffer significantly (t=0.32; p=0.75). The combined means of TG and GG(FIG. 2A) were significantly lower than the mean of TT (t=2.97;p=0.003). This suggests a dominant effect of the G allele over the Tallele. The difference between the means of drinks per drinking day inG-carriers and TT genotypic group was 2.59+0.87 (95% CI −0.879 to4.297).

Estrogen has been shown to modulate the synthesis, release, andmetabolism of 5-HT (Bethea et al., 2002; Frackiewicz et al., 2000; Pivacet al., 2004). Thus, to examine the impact of gender on theseassociations, we repeated the analyses on male subjects only. InCaucasian males, the mean difference of standard drinks per drinking daybetween G-carriers and TT genotypic group, was 2.89+1.07 (95% CI −0.771to 5.009), which was similar to the mean difference in combinedCaucasian male and female subjects. Therefore, we did not find asignificant effect of gender on the associations between rs1042173genotypes and drinks per drinking day.

However, among Hispanic subjects, we did not detect a significant effectof rs1042173 genotypes on both measures of drinking intensity, drinksper drinking day (F=0.935; p=0.397) and drinks per day (F=0.299;p=0.74).

Considering that 5′-HTTLPR L/S polymorphism has implicated as functionalin many reported studies, we examined potential interactive effect of5′-HTTLPR L/S and rs1042173 alleles on drinking intensity by using anewly developed algorithm for detecting gene-gene interaction, calledgeneralized multifactor dimensionality reduction (GMDR) method (Lou etal. 2007). Our GMDR analyses revealed no significant interaction betweenthese two functional SNPs (P=0.623).

5-HTT mRNA Expression in Cells Transfected with Plasmid Carrying EitherT or G Alleles

To study whether the T and G alleles of rs1042173 leads to differentialexpression levels of 5-HTTs, we transfected plasmids carrying the T andG alleles of rs1042173 into HeLa cells and quantified mRNA levels byusing the qRT-PCR assay. Results were analyzed for allelic differencesusing the ΔΔCt method described by Winer et al (1999). FIG. 3A depictsthe mean 5-HTT mRNA expression levels for the T and G alleles from threeindependent transfection experiments. The G allele yielded significantlyhigher mRNA expression level compared with the T allele. In the threeindependent experiments, the G allele-transfected HeLa cells, comparedwith their T allele-transfected counterparts, always produced a >50%higher 5-HTT mRNA level, an effect that was significant statistically(p<0.0001).

5-HTT Protein Expression in the T and G Alleles of the rs1042173 SNP

To determine if the allele-associated RNA difference can be translatedinto protein, we measured allele-specific differences in 5-HTT proteinlevels between the two alleles. After normalization with tubulin for theloading difference, we found that the 5-HTT protein level with G allele(0.137±0.006) is significantly higher than that of T allele(0.104±0.002) (t=5.53; p=0.005; See FIG. 3B). These results werereproduced in western blotting experiments from several independentreplications. Notably, the expression of both mRNA and 5-HTT proteinswas in the same direction the G allele being associated with higher mRNAand protein expression levels than the T allele.

Discussion

The data provide evidence that rs1042173, a SNP in the 3′UTR of theSLC6A4 gene, is associated with intensity of drinking among Caucasiansdependent on alcohol. Using a site-directed mutagenesis approach, it wasshown that rs1042173 is a functional polymorphism that resulted in adifference in 5-HTT expression levels in HeLa cell cultures, with Gallele associated with higher 5-HTT mRNA and protein expression levelsthan the T allele. Of multiple approaches used to determine whether apolymorphism is a function one, a direct comparison of expression levelbetween two alleles through an in vitro expression system as used inthis study represents one of the most convenient molecular techniques inthe field.

Alcohol-dependent individuals who were G-allele carriers for rs1042173showed less intensity of drinking compared with those who werehomozygous for the T allele. Importantly, the average intensity ofdrinking for both of these allelic groups exceeded the threshold forheavy drinking (i.e., ≥5 and ≥4 standard drinks/day for men and women,respectively), and all were dependent on alcohol. At the time of entry,subjects in both allelic groups were not statistically significantlydifferent in average chronological age and duration of alcoholdependency. It is, therefore, reasonable to propose thatalcohol-dependent individuals with the TT genotype might constitute asubtype of more intense drinkers among heavy-drinking alcoholics ofEuropean descent.

This is the first study to investigate the function of the rs1042173 SNPin an alcohol-dependent population. The rs1042173 polymorphism is notonly located at a putative polyadenylation signal site in the 3′UTR ofthe 5-HTT gene but also near a potential binding site for microRNAmiRNA-135 according to a bioinformatics prediction with PicTar program(Chen et al., 2006). It has been hypothesized that a variant at thislocation may change expression levels by affecting the stability of mRNA(Battersby et al., 1999; Beaudoing et al., 2000; Chen et al., 2006). Ourfindings have been further supported by two recent reports. The firststudy reported by Vallender et al. (2008) revealed that a functionalhaplotype containing T allele of rs1042173 was associated with highermRNA expression in HEK293 cells compared to the haplotype consisting ofG allele. Another study reported by Lim et al. (2006) showed thatG-allele had increased allelic expression imbalance (AEI) inEpstein-Barr virus transformed lymphoblast cells while human pons tissueshowed a decreased AEI for G-allele. Although the expression levelsassociated with each allele of rs1042173 are inconsistent among thesestudies (likely due to different reporter genes and/or cell lines usedamong them), they all reveal that rs1042173 is a functional one.

The finding of no association between rs1042173 genotype and intensityof drinking in Hispanics, which differed from that of an associationamong Caucasians, while the allelic frequencies for T and G alleles inCaucasians and Hispanics were not significantly different, does suggestthe possibility of differential regulation of gene expression by ethnicgroup. Due to the relatively small sample size of the cohort, such apremise needs to be treated as preliminary and confirmed by largerstudies.

The data show that the association between the intensity of drinking andthe genotype remained significant in the same manner even if we variedthe drinking period prior to enrollment within a range of 14 to 90 days(data not shown). The consistency of these results strengthened ourfindings.

The findings suggest the possibility that two different subgroups oftreatment-seeking alcoholics with allelic differences at the 3′UTR, SNPrs1042173 can differ in their intensity of drinking, an effect thatmight be associated with underlying differences in expression of 5-HTT.

TABLE 1 Biological Information of the 5 SNPs Examined in the Studyp-values for the deviation Primers and probe MAF from HWE^(b)sequences/context NCBI Physical Chromosome Cauca- His- Cauca- His-sequence ID of ABI dbSNP ID position position Alleles CEU^(a) sian^(b)panic^(b) sian panic primers and probes 5-HTTLPR Promoter ~25,588,500 L— 0.451 0.430 0.814 0.624 Forward: (long) TCCTCCGCTTTG S GCGCCTCTTCC(short) (SEQ ID NO: 13) Reverse: TGGGGGTTGCAG GGGAGATCCTG(SEQ ID NO: 14) rs25531 Promoter  25,588,472 A/G 0.100 0.065 0.079 0.9991.000 Forward: TCCTCCGCTTTG GCGCCTCTTCC (SEQ ID NO: 15) Reverse:TGGGGGTTGCAG GGGAGATCCTG (SEQ ID NO: 16) The two alleles were determinedusing restriction enzymes Hpa11 and bcc1 rs6354 Exon 2  25,574,024 T/G0.295 0.202 0.158 0.319 1.000 C_1841706_10 (5′ UTR) rs6355 Exon 3 25,572,936 C/G 0.025 0.022 0.026 1.000 1.000 C_11414113_20 (Ala/ Gly)rs28914832 Exon 10  25,562,500 A/C 0.008 0.008 0.009 1.000 1.000[Custom Taqman(R) (Leu/ SNP Genotyping Ile) Assay] Forward: GCAGAAGCGATAGCCAACATG (SEQ ID NO: 17) Reverse: CAAGCCCAGCG TGATTAACATC(SEQ ID NO: 18) Probe: CTTTCTTTGCC [C/A]TCATCT (SEQ ID NO: 19) rs1042173Exon 15  25,549,137 G/T 0.433 0.419 0.455 0.138 1.000 C_7473190_10 (3′UTR) MAF, minor allele frequency; HWE, Hardy-Weinberg equilibrium; ABI,Applied Biosystems (Foster City, CA). ^(a)European sample from HapMapproject. ^(b)Data from this study.

TABLE 2 Demographics and Drinking Parameters in the Cohort Analyzed forrs1042173 Hispanic Caucasian p- TT TG GG p-value TT TG GG value Numberof 47   77   41   — 26   56   28   — subjects Gender (% male) 82.9764.93 80.49 — 88.46 85.71 82.14 — Age (years)  41.6 ± 1.66 42.36 ± 1.2340.98 ± 1.52 0.62 37.08 ± 2.01 40.05 ± 1.22 38.82 ± 1.76 0.33 Age ofonset of 29.74 ± 1.72 30.61 ± 1.25 28.37 ± 1.87 0.69 26.44 ± 1.67 26.82± 1.23 26.36 ± 1.87 0.91 problem thinking Baseline drinks 11.17 ± 0.98 8.05 ± 0.47  9.58 ± 0.67 0.0043  9.99 ± 0.71 10.66 ± 0.67  9.76 ± 0.580.65 per drinking day Baseline drinks  8.99 ± 0.96  6.48 ± 0.44  7.72 ±0.58 0.02  8.23 ± 0.75  7.52 ± 0.59  7.92 ± 0.55 0.74 per day Years oflifetime 11.86 ± 1.32 11.75 ± 1.04 12.6 ± 1.4 0.56 10.63 ± 1.68 13.23 ±1.2  12.64 ± 1.51 0.35 drinking Values are means ± SEM. Significantp-values after correction for multiple testing are given in bold. “Yearsof lifetime drinking” was calculated by subtracting the age at which thesubject began experiencing symptoms of alcohol dependence from their ageat study enrollment. The adjusted p-value at the 0.05 significance levelis 0.010.

Bibliography for Example 1

-   American Psychiatric Association (1994) Diagnostic and statistical    manual of mental disorders, 4th ed. American Psychiatric    Association, Washington, D.C.-   Babor T. F., de la Fuente J. R., Saunders J., Grant M. (1992) AUDIT:    The alcohol use disorders identification test, World health    organization, Geneva, Switzerland.-   Barrett et al., Bioinformatics, 2005, 21:263-265.-   Battersby et a., J Neurochem, 1999, 72:1384-1388.-   Beaudoing et al., (2000), Genome Res 10:1001-1010.-   Bethea et al. (2002), Front Neuroendocrinol 23:41-100.-   Bradley et al. (1997), J Neurochem 69:1356-1367.-   Cargiulo T. (2007) Am J Health Syst Pharm 64(5 Suppl 3):S5-11.-   Chen et al. (2006) Hum Genet 120:1-21.-   Chen et al. (2006) Nature Genetics 38: 1452-1456-   Dundon et al. (2004) Alcohol Clin Exp Res 28(7):1065-73.-   Feinn et al., (2005), Am J Med Genet B Neuropsychiatr Genet    133B(1):79-84.-   Frackiewicz et al., (2000), Ann Pharmacother 34:80-88.-   Gastfriend et al., (2007), J Subst Abuse Treat 33(1):71-80.-   Goldman et al. (2005) Nature Reviews/Genetics 6:521-532.-   Gill K, Amit Z. (1989) Recent Dev Alcohol 7:225-48.-   Heils et al. (1996) J Neurochem 66:2621-2624.-   Hu et al. (2005) Alcohol Clin Exp Res 29(1): 8-16.-   Hu et al. (2006) Am J Hum Genet 78(5):815-26.-   Johnson et al. (2004) Alcohol Clin Exp Res 28(2):295-301.-   Javors et al. (2005) Prog Neuropsychopharmacol Biol Psychiatry    29(1):7-13.-   Johnson et al. (2008) Can serotonin transporter genotype predict    serotonergic function, chronicity, and severity of drinking? Prog    Neuropsychopharmacol Biol Psychiatry 32(1):209-16.-   Kweon Y S, Lee H K, Lee C T, Lee K U, Pae C U, (2005) Association of    the serotonin transporter gene polymorphism with Korean male    alcoholics. Journal of Psychiatric Research 39:371-376.-   LeMarquand et al. (1994) Biol Psychiatry 36:326-337.-   Lim et al. (2006) Allelic expression of serotonin transporter (SERT)    mRNA in human pons: lack of correlation with the polymorphism    SERTLPR. Mol Psychiatry 11(7): 649-662.-   Little et al. (1998) Am J Psychiatry 155:207-213.-   Lou et al. (2007) Am J Hum Genet 80(6):1125-1137.-   Makela P. Mustonen H. (2007) Alcohol Alcohol 42(6):610-7.-   Mynett-Johnson et al. (2000) Am J Med Genet 96(6):845-9.-   Ozaki et al. (2003) Mol Psychiatry 8(11):933-6.-   Pivac et al. (2004) Life Sci 76:521-531.-   Prasad et al. (2005) Human serotonin transporter variants display    altered sensitivity to protein kinase G and p38 mitogen-activated    protein kinase. PNAS 102(32):11545-11550.-   Qian et al. (1997) Protein kinase C activation regulates human    serotonin transporters in HEK-293 cells via altered cell surface    expression. J Neurosci 17:45-57.-   Ramamoorthy et al. (1993) Antidepressant- and cocaine-sensitive    human serotonin transporter: molecular cloning, expression, and    chromosomal localization. Proc Natl Acad Sci USA 90:2542-2546.-   Sullivan et al. (1989) Br J Addict 84:1353-1357.-   Talvenheimo et al. (1980). J Biol Chem 255:8606-8611.-   Vallender et al. (2008) Functional variation in the 3′ untranslated    region of the serotonin transporter in human and rhesus macaque.    Genes, Brain Behav. 2008 August; 7(6):690-7-   Wendland et al. (2006) Simultaneous genotyping of four functional    loci of human SLC6A4, with a reappraisal of 5-HTTLPR and rs25531.    Mol Psych. 11:224-226.-   Winer et al. (1999) Anal Biochem 270:41-49.-   Wrase et al. (2006) Cogn Affect Behav Neurosci 6:53-61.

Example 2 Drinking Histories in Alcohol-Use-Disordered Youth:Relationship of Platelet Serotonin Transporter Expression with Genotypesof the Serotonin Transporter

Functional control of the serotonin system is hypothesized to beregulated in part by differences in SERT (5-HTT) expression [15]. Thegene responsible for encoding SERT expression has a functionalpolymorphism at the 5′-regulatory promoter region [16, 17]. Thepolymorphism contains an insertion/deletion mutation with the long (L)variant having 44 base pairs that are absent in the short (S) variant.In normal controls, the LL genotype, compared to S-carriers (i.e., SSand SL genotypes), has greater 5-HT uptake in human platelets [18],lymphoblasts [19], and greater numbers (e.g., reflecting either greaterexpression or less turnover) of SERT in human raphe nuclei [20].Assuming that individuals with the LL genotype have greater SERTexpression rates, these individuals would be hypothesized to havegreater 5-HT uptake, lower intra-synaptic 5-HT levels, and, therefore,reduced intra-synaptic 5HT neurotransmission in vivo and in vitro [16,19].

Differential expression of the serotonin transporter, in interactionwith chronic alcohol use, may play an important role in the etiology andpathogenesis of alcoholism [15, 21], especially for early-onsetalcoholism. For example, adolescents with the LL genotype may havespecific vulnerabilities that increase the risk of developing alcoholism[1, 22]. Family risk and population studies provide support for thishypothesis. In a sample of men at risk for alcohol dependence, the LLgenotype was more prevalent among those who developed alcohol dependence[23]. Ernouf and colleagues [24] have shown that platelet serotonin(5-HT) uptake was higher in alcohol dependent parents and theirchildren, compared to age-matched controls. Rausch and colleagues [25]showed that adult males with alcohol-dependent fathers had higher meanVmax for platelet 5-HT uptake, compared to FH-controls. In a Japanesesample, alcoholics with the L allele (e.g., LL and LS genotypes) had asignificantly earlier onset of alcohol dependence, compared those withthe SS genotype [26]. In a Korean male sample, the frequency of theL-allele was significantly higher in alcohol dependent individualscompared to controls [27]. However, in European and Mexican-Americansamples, the SS genotype, not the LL genotype, has been associated withan antisocial-type of alcoholism [28, 29], so the genetic risk isclearly not as simple as a single allelic variant increasing risk foralcohol dependence. Our group has reported previously that 5-HT uptakeinto platelets was greater among EOA males, compared to LOA males andhealthy controls [30]. Although in adult normal samples, platelet 5-HTuptake is greater among L-carriers compared to individuals with the SSgenotype [18], we recently found that among adults with chronic alcoholdependence, both 5-HT uptake and 3H-paroxetine binding to SERT werereduced among L-carriers (e.g., LL and LS) compared to SS homozygotes[31], and these reductions in platelet 5-HT function were related toyears of drinking. Together, the above findings from family risk andpopulation studies support the hypothesis that chronic alcohol use maybe “toxic” to SERT and diminish SERT activity expressed in individualswho are L-carriers [15, 20, 22].

The aims were to determine whether SERT genotype (LL vs. S-carriers)differentiated Early Onset adolescents with AUD with respect to theirdrinking patterns or their serotonergic activity measured by SERTdensity and function in platelets. Relationships of platelet SERTmeasures to current and lifetime drinking also were examined. It washypothesized that platelet measures of SERT binding and function wouldbe related to SERT genotype, in part due to the relatively shorthistories of alcohol use. Specifically it was predicted that adolescentswith AUD would show higher SERT function and SERT density in the LLgenotype compared to the S-genotypes (LS, SS). It was also testedwhether history of drinking, current amount of drinking, or both,determine how SERT genotype alters SERT function in adolescents withalcohol use disorder.

Materials and Methods

Participants—Participants were youths aged 18-20 with a current alcoholuse disorder who were not seeking treatment. Participants were diagnosedwith alcohol abuse or alcohol dependence, using criteria from theDiagnostic and Statistical Manual of Mental Disorders 4th edition(DSM-IV; American Psychiatric Association, 1994). All volunteers were ingood physical health (determined by a complete physical examination,electrocardiogram (EKG) within normal limits, and laboratory screeningtests within acceptable parameters); were consuming at least 3 StandardDrinks/Drinking Day; had breath-alcohol level of zero at screen; andwere literate in English. Exclusionary criteria included current orlifetime Axis I DSM-IV Substance Use Disorder other than to alcohol orcannabis use disorder. Illicit substance use in the past 30 days otherthan marijuana use was exclusionary. Psychiatric exclusionary criteriaincluded current major depressive disorder, bipolar disorder,post-traumatic stress disorder, psychosis, or attention deficithyperactivity (ADHD) that was medicated within the previous 30 days.Medical exclusionary criteria included elevated liver enzymes greaterthan 4 times the normal range, and/or elevated bilirubin (>110% perlimits of normal); serious medical co-morbidity requiring medicalintervention or close supervision; clinically significant alcoholwithdrawal; treatment for alcohol abuse or dependence within the last 30days; or, if female, pregnant. All participants gave written informedconsent. The study was approved by the University of Texas HealthScience Center Institutional Review Board.

Diagnostic Measures—Trained therapists used the Children's interview forPsychiatric Syndromes (ChIPS), which adheres strictly to DSM-IV criteriafor psychiatric disorders and has been shown to be accurate and providevalid diagnoses of psychiatric disorders in adolescents and young adultsto 20 years of age [32]. Current and lifetime substance use disorderswere diagnosed in a structured clinical interview using the AdolescentDiagnostic Interview (ADI) [33, 34]. The principal investigatorclarified discrepancies and established reliability of interviews, usingthe Best Estimate Diagnostic Procedure [35, 36], and monitored forconsistency of participant's self-report throughout the study. Recentreported drinking and lifetime drinking were determined by patientinterview using time-line follow back procedures [37].

General design and procedures—The experimental design was across-sectional retrospective study. After initial screening, eligibleparticipants gave blood for assay of platelet 5HT function andgenotyping. Fifty milliliters of blood was drawn from each participantto obtain platelets for the measurement of 5-HT uptake into intactplatelets and paroxetine binding to platelet membranes. Additionally a10 ml sample of blood was drawn for the determination of SERT genotype.

Platelet Suspension and Platelet Membrane Preparation—Fifty ml blood wasdrawn into 60 ml polypropylene syringes containing 10 ml ofAcid-Citrate-Dextrose (ACD) buffer. The blood was then centrifuged at150 g at 23° C. for 20 min in a Beckman TJ-6 centrifuge to obtainplatelet-rich plasma (PRP). Platelet count in PRP was determined with aCoulter counter model S-plus VI and adjusted to 3×108 platelets/ml withthe addition of platelet buffer (137 mM KCl, 1 mM MgCl₂. 5.5 mM glucose,5 mM HEPES, pH 7.4) to prepare adjusted PRP for the serotonin uptakeexperiments only. Three ml of adjusted PRP was used for plateletserotonin uptake experiments, which were performed on the day of theblood draw. To prepare platelet membranes for paroxetine bindingexperiments the remainder of the PRP was used. One ml of prostaglandin12 solution (300 ng/ml) per ml of PRP was added to prevent loss ofplatelets during centrifugation, and then the sample was centrifuged at550 g. The resulting platelet pellet was resuspended in platelet bufferand then centrifuged at 35,000 g. The platelet membrane pellet wasresuspended in 1 ml of platelet buffer and then stored at 80° C. untilthe day of the assay to measure paroxetine binding.

Serotonin Uptake into Intact Platelets—Platelet 5-HT uptake experimentswere performed in 21 participants. The adjusted PRP suspension was usedto determine platelet 5-HT uptake. Assay tubes were prepared induplicate and contained ³[H] 5-HT at six different concentrations (62.5nM to 2000 nM), and 100 μM pargyline with or without 50 μM fluoxetine.These tubes were incubated at 37° C. for 5 min; then the reaction wasstarted by the addition of 100 μl of adjusted PRP that contained 107platelets. The assay tubes were incubated at 37° C. for an additional 5min; then the reaction was quenched by rapid filtering through WhatmanGF/B filters using a Brandel Cell Harvester. The filters were washedthree times with 5 ml of ice-cold wash buffer (50 mM Tris-HCl, 150 mMNaCl, and 20 mM ethylene diamine tetra-acetic acid (EDTA)). Filters wereplaced in scintillation vials containing 5 ml of Beckman Ready Protein+scintillation counting fluid and immediately counted. Specific uptakewas calculated by subtracting total uptake front nonspecific uptake(fluoxetine tubes). Maximum 5-HT uptake rate (Vmax) in platelets wasexpressed as fmol 5-HT/min-10⁷ platelets, and the equilibrium constant(Km) as nM. Km and Vmax were calculated using the one-site hyperbolicfunction in Prism 4 software by Graph Pad™.

Paroxetine Binding to Platelet Membranes—Platelet membranes were used todetermine platelet paroxetine binding. Assay tubes were prepared induplicate containing incubation buffer (50 mM Tris-HCl, 5 mM KCl and 120mM. NaCl) and ³[H] paroxetine at 6 different concentrations (0 to 2 nM)with and without 150 mM fluoxetine. The actual concentration ofparoxetine in each tube was determined using a 40 ml aliquot taken fromeach tube prior to the addition of platelet membranes. The experimentwas started by the addition of 80 mg of platelet membrane protein thenthe assay tubes were incubated for 1 hr at 23° C. The reaction wasquenched by addition of ice-cold wash buffer (50 mM Tris HCl, 150 mMNaCl, 20 mM EDTA) and rapid filtering through Whatman GF/B filterstreated with 0.3% polyethylenimine using a Brandel Cell Harvester.Filters were washed 3 times with ice-cold wash buffer, dried over-night,placed in scintillation vials containing 5 ml of Beckman Ready Protein+scintillation counting fluid and counted in a Beckman LS-6500 liquidscintillation counter. Disintegrations per minute (DPM) from the 40 mlaliquots were converted into nM of paroxetine to obtain the actualconcentrations in each tube. Total and non-specific binding ofparoxetine was plotted against each actual concentration. Specificbinding was calculated by subtracting non-specific binding from totalbinding. Kd and Bmax of paroxetine binding were calculated using Prism4software (Graphpad). Paroxetine binding (Bmax) was expressed as fmol/mgof platelet membrane protein and Kd as nM. Protein concentrations weremeasured using the BioRad method and a SPECTRAmax PLUS384 Micro-platespectrophotometer.

Genotyping—The blood sample for the determination of SERT genotype wasdrawn at enrollment. White blood cells were separated from plasma andre-suspended, and DNA was isolated using PUREGENE, Gentra systemsaccording to the manufacturer's protocol. The 5′-HTTLPR 44 bp promoterregion repeat polymorphism was amplified by polymerase chain reaction(PCR) from ˜50 ng of DNA using two primers: 5′-CGT TGC CGC TCT GAA TGCCAG-3′ (SEQ ID NO:11) AND 5′-GGA TTC TGG TGC CAC CTA GAC GCC-3′ (SEQ IDNO:12) and in a 25-ul final volume consisting of 0.5 U of Tfl DNApolymerase (Epicentre), 1× PCR buffer, 1.5 ml MgCl₂, 200 uM dNTPs, lxenhancer, and 0.6 uM of each primer. The PCR conditions were as follows:94° for 30 s; 70° C. for 30 s, and 72° C. for 30 s); a final extensionof 72° C. for 7 min and terminal hold at 4° C. Separation by gelelectrophoresis using 4% MetaPhor agarose (Cambrex, Rockland, Me.)allowed visualization by ethidium bromide/UV detection of the twovariants (long (L) and short (S): fragment sizes=464 bp and 420 bp,respectively) of the promoter region of the SCL4A gene (−1415 to −951)[16].

Statistical Analyses—Means and standard deviations for outcome variablesof platelet variables (Km and Vmax of 5HT uptake into intact platelets,and Kd and Bmax of paroxetine binding on platelet plasma membranes) wereexamined. Non-normal distributions of outcome variables weretransformed. Planned analyses included Pearson correlations to examinethe relationships the platelet variables. T-tests were used to determinewhether there were group differences in the LL genotypes vs. S-carriergenotypes, in psychiatric disorders, current, and lifetime drinking, forthe dependent variables for platelet 5HT function (Bmax Kd. Vmax, andKM).

Results—

The distribution of genotypes was the following: LL, n=8, LS, n=9, SS,n=4. Since S-carriers (LS and SS) were the predominant genotypes in thesample, LS and SS were pooled for the analyses of group differences(e.g., LL vs. S-carriers).

There were no statistically significant differences in age or ethnicitybetween the LL and S-carriers (see Table 1). However, the LL group had asignificantly earlier age of onset and longer duration of alcohol use,but did not have significant differences in quantitative measures ofrecent drinking. The LL group also had significantly higher inattentionand motor components of trait impulsivity, and a trend towardssignificant differences in total BIS-11 trait impulsivity. Allparticipants had a current Alcohol Use Disorder based on DSM-IVcriteria. There were no significant differences between genotype groupsin other the DSM-IV psychiatric groups.

Table 2 and FIG. 4 present the results of the platelet studies.Participants having the LL genotype had significantly higher Bmax and Kdthan did S-carriers, indicating greater amounts of SERT with loweraffinity for paroxetine binding. There were no genotype groupdifferences in the platelet functional measures of 5HT uptake.

Discussion—

The main finding was that SERT genotype predicted differences in age ofonset and duration of drinking, as well as the platelet binding profileof SERT among adolescent subjects with an alcohol use disorder.Specifically, adolescents with the LL genotype began drinking at ayounger age and showed greater 3H-paroxetine binding at lower affinitythan did S-carriers.

Even though both groups of participants had an onset of alcohol usedisorder during adolescence and were of the same current age (i.e.,mean=18.7 yrs), participants with an LL-genotype had significantlyearlier age of onset of drinking (i.e., 13.5 vs. 15.2 years of age)compared to the S-carriers. This finding is consistent with a hypothesisdescribed by Johnson [22] predicting that the LL genotype would beassociated with an earlier age of onset of problem drinking. Alsoconsistent with the Johnson hypothesis, the LL-group also had higherlevels of behavioral vulnerability (i.e., trait impulsivity) than didthe S-carriers [1, 15, 22]. This latter finding is interesting in lightof the literature suggesting LL-genotypes may have lower 5-HT in thesynaptic cleft and lower central turnover of 5-HT associated with higherlevels of impulsivity [1, 15, 22]. Interestingly, there were nosignificant group differences in the current levels of drinking, whichcontrasts with reports in college students, showing that S-carriers hadheavier patterns of binge drinking [38].

Results front previous studies in adult populations have generally shownthat the SS genotype is associated with antisocial types of alcoholismin European and Mexican-American populations [28], while among Asianpopulations, LL-genotype has been associated with alcoholism risk [26,29]. The present study population of adolescents included Caucasian,“white”—Hispanic, biracial or mixed, and American Indian ancestryparticipants. The results suggest that the LL genotype may be associatedwith a greater impulsivity, thereby increasing risk to beginencountering problem drinking at an earlier (adolescent) age. Given theassociation of the SS-genotype with anxiety and stress-related disorders[15, 39], an alternative hypothesis is that by the time that Caucasianadolescents mature into college-age young adults, other environmentalfactors such as stress, interact with S-carrier genotypes, to produceanxiety or affective distress resulting in greater patterns of drinkingand alcoholism risk.

Previous studies in adults have shown that compared to S-carriers, theLL-genotype is associated with increased central SERT binding [20] andincreased 5-HT uptake (but not binding) in the platelets of healthysubjects [18]. However, this finding appears not to be the case in adultalcoholics. It is known that adult alcoholics having an L-alleleactually have reduced 3H-paroxetine binding and 5-HT uptake intoplatelets compared to the SS-homozygotes—and hypothesized that thiseffect is related to years of problem drinking [31]. The currentfindings suggest that adolescent problem drinkers who have the LLgenotype initially have normal patterns of increased SERT binding, butthat S-carriers do not have normal SERT binding. Therefore, it isreasonable to speculate that with continued heavy drinking, adolescentswho have earlier onset of drinking and longer duration of drinking havean earlier onset of down regulation of SERT than is seen in adultalcoholics.

Example 2,

TABLE 1 Demographics, Drinking History, and Current PsychiatricDisorders Genotype LL LS/SS (n = 8) (n = 13) Variable Mean (SD) Mean(SD) P value Age (yrs) 18.9 0.6 18.5 0.5 0.20 Trait Impulsivity (BIS-11)Non-planning 26.0 4.7 24.4 6.9 0.57 Inattention 20.5 3.4 16.5 3.8 0.03Motor 27.8 3.2 23.9 3.2 0.02 Total 74.3 7.4 64.9 11.3 0.05 LifetimeDrinking Age of Onset of Alcohol Use (yrs) 13.5 1.2 15.2 1.9 0.03Duration of Alcohol Use (yrs) 5.4 0.9 3.3 1.8 <0.01* Recent Drinking DD3.0 1.7 3.9 5.3 0.98 DDD 9.9 5.7 7.8 7.8 0.27 PDA 67.6 16.1 52.7 25.70.16 (Percent of (Percent of LL LS/SS Number Participants) NumberParticipants) Gender 0.97 Male 5 62.5 8 61.5 Female 3 37.5 5 38.5Ethnicity 0.38 Caucasian 2 25.0 3 23.1 Hispanic 3 37.5 9 69.2 Biracialor Mixed 3 37.5 0 0.0 American Indian 0 0.0 1 7.7 ADHD 3 37.5 3 25.00.48 ODD 1 12.5 3 25.0 0.55 CD 6 75.0 9 75.0 0.92 Mood Disorders 2 25.03 23.0 0.85 Anxiety Disorders 0 0.0 4 44.0 0.81 Alcohol Use Disorder 8100.0 13 100.0 + Alcohol Dependence 8 100.0 10 83.3 0.14 Alcohol Abuse 00.0 3 16.7 ** Cannabis Dependence 1 12.5 6 50.0 0.11 *Duration ofalcohol use remains significant after including Barrett ImpulsivityScale (BIS) total as covariate. DD: Average Drinks per Day in past 90days; DDD: Average Drinks per Drinking Day in past 90 days; PDA: PercentDays Abstinent in past 90 days; + All participants met criteria for acurrent Alcohol Use Disorder; ** Three participants met DSM-IV-TRcriteria for Alcohol Abuse. Attention Deficit Hyperactivity Disorder(ADHD); Oppositional Defiant Disorder (ODD), Conduct Disorder (CD)

Example 2

TABLE 2 Group differences in measures of 5HT uptake and paroxetinebinding Genotype LL LS/SS (n = 8) (n = 13) Variable Mean (SD) Mean (SD)P value Paroxetine Binding B_(max) (fmol/mg protein) 802.0 254.2 504.3199.8 0.02 K_(d) (nM) 0.7 0.5 0.4 .3 0.03 Bmax/Kd 1293.2 508.6 1861.41318.0 0.18 5HT Uptake Vmax (fmol/min−10⁷ 181.6 128.4 200.1 113.5 0.46platelets) K_(m) (μM) 445.9 409.3 323.2 136.4 0.40 V_(max)/K_(m) 0.6 0.40.7 0.4 0.53 Note: Data were transformed to the natural log scale fort-test analyses.

Conclusion—

The present findings provide partial support for the hypothesis thatamong currently drinking adolescents with an alcohol use disorder, thosehaving a LL-genotype display greater impulsivity, began drinking at anearlier age, and have increased ³H-paroxetine binding to platelet SERT.These findings expand our current understanding of the 5′-promoter ofthe SERT gene in regulating the SERT in adolescents with AUD. This studyprovides preliminary findings that further demonstrate that platelet andgenetic measures of SERT function may be useful measures to track thecomplex interplay of biological and environmental factors in theetiology of vulnerability and risk of either alcoholism onset ortoxicity.

Example 2 Bibliography

-   1. Johnson, B. A., and N. Ait-Daoud, Psychopharmacology, 2000.    149: p. 327-344.-   2. LeMarquand et al., Biological Psychiatry, 1994. 36: p, 326-337.-   3. LeMarquand et al., American Journal of Psychiatry, 1999, 156: p.    1771-1779.-   4. Stoltenberg, S. F., Alcoholism: Clin. Exp. Res., 2003. 27: p.    1853-1859.-   5. Linnoila et al., Life Sciences, 1983. 33: p. 2609-2614.-   6, Fils-Aime, M. L., et al., Archives of General Psychiatry, 1996.    53(3): p. 211-216.-   7. Cloninger, C., Science, 1987. 236: p. 410-416.-   8. Virkkunen et al., Archives of General Psychiatry, 1987. 44: p.    241-247.-   9. Virkkunen et al., Archives of General Psychiatry, 1996. 53: p.    523-529.-   10. Swann et al., Psychopharmacology, 1999. 143: p. 380-384.-   11. Grunbaum, J. A., et al., Morb. Mort. Wkly Rpt., Surveil.    Sum. 2002. 51(4): p. 1-62.-   12. McBride, et al., Critical Reviews in Neurobiology, 1998. 12: p.    339-369.-   13 Virkkunen, et al., Journal of Psychiatry and Neuroscience, 1995.    20: p. 271-275.-   14. Virkkunen, et al., Epidemiology, Neurobiology, Psychology,    Family Issues., M. Galanter, Editor. 1997, Plenum Press: New    York. p. 173-189.-   15. Heinz et al., Psychopharmacology, 2004. 174: p. 561-570.-   16. Heil et al., Journal of Neurochemistry, 1996. 66: p. 2621-2624.-   17. Heils et al., Journal of Neural Transmission, 1997. 104: p.    1005-1014.-   18. Greenberg et al., American Journal of Medical Genetics, 1999,    88: p. 83-87.-   19. Lesch, et al., Science, 1996. 274: p. 1527-1531.-   20. Heinz, et al., Biological Psychiatry, 2000. 47: p. 643-649.-   21. Meltzer, et al., Psychiatry Research, 1998. 24: p. 263-269.-   22. Johnson, B. A., et al., Alcoholism: Clin. Exp. Res., 2000,    24(10): p. 1597-1601.-   23. Schuckit, et al., Biological Psychiatry, 1999. 45: p. 647-651.-   24. Ernouf, et al., Life Sciences, 1993. 52: p. 989-995.-   25. Rausch, J. L., et al., Neuropsychopharmacology, 1991. 4(2): p.    83-6.-   26. Ishiguro, et al., Alcoholism: Clin, Exp. Res., 1999. 23: p.    1281-1284.-   27. Kweon, et al., Journal of Psychiatric Research, 2005. 39: p.    371-376.-   28. Feinn, et al., American Journal of Medical Genetics Part B    (Neuropsychiatric Genetics), 2005. 133B: p. 79-84.-   29. Konishi, et al., Alcohol, 2004. 32: p. 15-52.-   30. Javors, et al., Alcohol and Alcoholism, 2000. 35: p. 390-393.-   31. Johnson, et al., Neuropsychopharmacology and Biological    Psychiatry, in press.-   32. Rooney, et al., Administration manual of the ChIPS, 1999,    Washington, D.C.: American Psychiatry Press.-   33. Winters, et al., Adolcs. Diagnostic Interview Schedule and    Manual. 1993, Los Angeles: Western Psychological Services.-   34. Winters, K. C., et al., Psychology of Addictive Disorders, 1993.    7: p. 185-196.-   35. Leckman et al., Archives of General Psychiatry, 1982. 39: p.    879-883.-   36. Kosten, et al., American Journal of Psychiatry, 1992. 149: p.    1225-1227.-   37. Sobell, L. C., Sobell, M. B., Timeline follow-back: A technique    for assessing self-reported alcohol consumption, in Measuring    Alcohol Consumption: Psychosocial and biochemical methods, E. R.    Litten, Allen, J., Editor. 1992, Humana Press Inc.: Totwa, N.J. p.    41-72.-   38. Covault et al., Biological Psychiatry, 2007. 61(5): p. 609-16.-   39. Lesch, K. P., European Journal of Pharmacology, 2005. 526: p.    113-124.-   40. Dawes, et al., Alcohol and Alcoholism, 2004. 39(3): p. 166-177.-   41. Pine, et al., Archives of General Psychiatry, 1997. 54: p.    839-846.-   42. Soloff et al., Alcoholism: Clin. Exp. Res., 2000, 24(11): p.    1609-1619.-   43. Twitchell et al., Alcoholism: Clin. Exp. Res., 2000. 24(7): p.    972-979.-   44. Twitchell, et al., Alcoholism: Clin. Exp. Res., 2001. 25(7): p.    953-959.

Example 3 LL Alcoholics Experience Greatest Reduction in DrinkingSeverity Following Ondansetron Treatment

5-HT3 up-regulation increases the function of DA (Blandina et at 1989;Blandina et al 1988; De Deurwaerdere et al 1998), the principalneurotransmitter mediating alcohol's rewarding effects. Thisup-regulation may be increased by bouts of binge drinking because theextent to which the 5-HT3 receptor is potentiated is inversely relatedto the level of basal 5-HT neurotransmission (Lovinger 1991; Levinger1999; Lovinger and Zhou 1994; Lovinger and Zhou 1998; Zhou and Lovinger1996; Zhou et al 1998). Ondansetron may, therefore, be differentiallyeffective in EOA with presumed LL variant predominance by blockade ofup-regulated 5-HT3 receptors, thereby ameliorating the serotonergicdysfunction and decreasing alcohol's rewarding effects.

Polymorphic variation of the SERT at 5′-HTTLPR also may explain thetherapeutic treatment response to SSRIs among type A alcoholics (similarto LOA) with presumed SS/SL predominance (Pettinati et al 2000). Thisassociation is, however, probably not mediated through 5-HT3 mechanisms.It is proposed herein that in Pettinati et al.'s type A alcoholics,predominantly with the SS/SL form, basal serotonergic function wasnormal. Chronic SSRI treatment, therefore, produced modest facilitationof 5-HT neurotransmission and long-term inhibition of dopaminergicactivity, thereby offsetting alcohol's rewarding effects during chronicdrinking. Individuals with the SS/SL form of 5′-HTTLPR can be expectedto experience a similar modest anti-rewarding effect during acutealcohol intake while receiving chronic SSRI treatment. In contrast,chronic SSRI treatment was probably ineffective at reducing theprotracted drinking of type B alcoholics (Kranzler et al 1996) withpresumed LL predominance because serotonergic activity would have beenincreased greatly (as there are relatively fewer SERT transporters inthis state), and the ensuing marked hypo-dopaminergic state probablytriggered relief drinking to normalize tins neurochemical condition.Chronic SSRI treatment probably has little effect on 5-HTneurotransmission among acutely drinking individuals with the LL variantbecause basal serotonin reuptake already is enhanced greatly.

The present studies were performed to determine if the effective ofondansetron treatment could be correlated with the expression of the LLvariant of 5′-HTTLPR and alcohol consumption.

Materials and Methods

In a pre-planned interim analysis, data were examined for the 226alcohol-dependent individuals (aged 18-65 years) enrolled into the12-week randomized controlled pharmacotherapy trial to determine theeffect of ondansetron on drinking among individuals who varied onallelic difference at the 5-HTT gene and age of alcoholism onset. Allthese individuals were enrolled at the University of Texas HealthScience Center at San Antonio. Briefly, the study design was 2 (LL vs.LS/SS)×2 (early onset vs. late onset)×2 (ondansetron 4 μg/kg b.i.d. vs.placebo). The inferential results below are for the severity ofdrinking—drinks/drinking day (DDD).

Demographic data were that: 74% were male and 26% female; 48% wereearly-onset alcoholics and 52% late-onset alcoholics, and 20% wereHispanic and 80% White. There were no significant differences (P>0.05)on demographics between the treatment groups. Baseline mean (SD) DDD(past 90 days) were also similar for the ondansetron 4 μg/kg b.i.d. andplacebo groups—9.83 (4.63) vs. 9.85 (4.49), respectively. Theinferential analyses were conducted on all randomized subjects accordingto the intent-to-treat principle. The analytic plan was first tocalculate the DDD in each week. We then used the difference betweenweekly DDD and the baseline DDD (in the past 90 days) as repeatmeasures. A mixed-model approach (SAS PROC MIXED) was used to study theeffect of treatment, genotype (LL vs. LS/SS), treatment and genotypeinteraction, age, age of onset (early vs. late), gender, and age atonset of problem drinking, adjusting for the baseline DDD level. Alsoincluded is a random slope for time to study the variation in the timetrend of the weekly DDD.

Results—Example 3

It was observed that DDD for both the ondansetron and placebo groups hada roughly linear decreasing time pattern; thus, all groups improvedtheir drinking outcomes over time (F=32.96; P<0.0001). The table below(Table 1—Example 3) shows the cell contrasts for the different genotypeson DDD for the placebo and treatment (i.e., ondansetron) groups.

There also was a main effect of treatment (F=5.64; P=0.02). Theinteraction of treatment and genotype was highly significant (F=6.99;P=0.0083). There also was a marginally significant effect of age ofonset (F=3.68; P=0.06). There was an overall significant effect for theLL group to reduce DDD (F=5.64; P=0.02) and an effect of time (F=12.69;P=0.0007). From the table, the cell contrasts show that the reduction inDDD for the LL group was driven by the fact that the ondansetron LLgroup had a significantly greater reduction in DDD compared with theother allelic types. Indeed, the effect size (Cohen's d) forondansetron's effect in LL individuals to reduce DDD was large (i.e.,0.08). The mean (SEM) DDD reduction from baseline across the treatmentperiod for the different genotypes and treatment conditions was 5.70(0.64) for ondansetron LL, 3.45 (0.44) for ondansetron LS/SS, 3.54(0.67) for placebo LL, and 4.25 (0.45) for placebo LS/SS. About 70% ofthose who entered the double-blind phase completed the trial.

These promising data provide the first evidence that alcoholics with theLL genotype, compared with their LS/SS counterparts, experiencesignificantly greater reduction in the severity of drinking followingondansetron treatment.

TABLE 1 Example 3 Lower Upper P- Cohen's Treatment Genotype Estimate CICI Value d Placebo LS/SS vs. −0.71 −2.29 0.87 0.379 0.03 LL TreatmentLS/SS vs. 2.25 0.73 3.78 0.004 0.10 LL Placebo vs. LS/SS −0.80 −2.030.43 0.203 0.04 treatment Placebo vs. LL 2.16 0.35 3.98 0.020 0.08treatment

Critically, these new findings on serotonergic medications revive theconcept that alcoholism is a heterogeneous disorder associated withvarying neurochemical abnormalities. Medications that specificallytarget one or more of these underlying abnormalities promise, therefore,to be powerful treatments, and their trials should advance ourscientific understanding of the disease.

Example 4 Methods of Predicting Responses to Treatment Based onDifferent Genotypes of the 5′-HTTLPR and the 3′UTR of the SerotoninTransporter Gene SLC6A4 and Methods of Treatment Based on theDifferences

Based on the results of the experiments described in Examples 1-3, aseries of studies were performed to determine whether there is apharmacogenetic effect of ondansetron to differentially treat those withthe LL genotype of the 5′-HTTLPR, the TT genotype of the 3′-UTR ofrs1042173, or the combination of the genotypes.

Materials and Methods

Subjects:

289 alcohol-dependent men and women enrolled in a 12-week treatmenttrial in which they received either ondansetron (4 μg/kg) or placebo.All subjects also received weekly cognitive behavioral therapy as theirstandardized psychosocial treatment. Genotyping was conducted on allsubjects.

Statistical Methods:

Mixed-effects models were used to study the effect of treatment andgenotype and their interaction for each of the primary drinkingoutcomes. The models included random intercept and random slope and wereadjusted for covariates such as participants' average 90-day drinkinglevels prior to the study, age, gender, ethnicity (Caucasian andHispanic), and center. A variance-components covariance matrix was usedto model different variances for the intercept and slope and acovariance between them. Interactions of treatment, genotype, age, andcenter were first included in the models. They were excluded from thefinal models if not significant.

Results:

For drinks per drinking day (DDD) outcome, it was found that there weresignificant rs1042173 main effects in DDD (p=0.003) as well as in thers1042173-by-5′-HTTLPR L/S alleles (LL, LS/SS) interaction effect(p=0.021) and the 5′-HTTLPR L/S alleles-by-treatment interaction effect(p=0.028). Patients with the TT genotype had more than a 1-DDD reductioncompared with those with TG/GG (mean difference=−1.16; 95% CI: −1.93 to−0.39; p=0.003). Unexpectedly, in patients with bath the LL and TTgenotypes (LT), the DDD reduction was much greater than in those withthe other genotype combinations of rs1042173 and 5′-HTTLPR L/S alleles(p<0.05), and there was more than a 2-DDD reduction in LT individualscompared with those who had LL and TG/GG (LG; mean difference=−2.06; 95%CI: −3.27 to −0.85; p=0.001). When treated with ondansetron, TT genotypepatients seemed to respond, to treatment more effectively than did TG/GGgenotype patients (mean difference=−1.31; 95% CI: −2.36 to −0.25;p=0.016). A similar treatment effect was observed when we comparedpatients with the LL genotype with those who had LS/SS (meandifference=−1.41; 95% CI: −2.46 to −0.36; p=0.009), and among patientswith the LL genotype, those in the treatment group had a 1.5-DDDreduction compared with those in the placebo group (meandifference=−1.50; 95% CI: −2.70 to −0.31; p=0.013). Similar effects wereobserved for other drinking measures.

Conclusion:

Ondansetron exerts a preferential treatment effect to reduce severedrinking among alcohol-dependent individuals with the LL genotype of the5′-HTTLPR, an effect that is increased among those who also possess theTT allele in the 3′-UTR of rs1042173. These data demonstrate animportant pharmacogenetic effect of ondansetron in alcohol-dependentindividuals. This study validates a method whereby alcohol-dependentindividuals identified as having either of these alleles, or theircombination, can be treated effectively with ondansetron.

The data presented in Examples 1 and 4 demonstrate that there is anassociation with higher severe drinking and susceptibility toondansetron treatment in alcohol-dependent subjects homozygous for T,relative to alcohol-dependent subjects with a G allele.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated by reference herein intheir entirety.

Headings are included herein for reference and to aid in locatingcertain sections. These headings are not intended to limit the scope ofthe concepts described therein under, and these concepts may haveapplicability in other sections throughout the entire specification.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

What is claimed is:
 1. A method of treating an opioid-related disorder,comprising: administering an antagonist of the serotonin receptor 5-HT3to a patient with an opioid-related disorder: wherein the patient isknown to have the LL genotype of functional polymorphism serotonintransporter-linked polymorphic region 5-HTTLPR of the serotonintransporter gene SLC6A4 and known to have the TT genotype of the singlenucleotide polymorphism rs1042173 of the serotonin transporter geneSLC6A4.
 2. The method of claim 1, wherein the 5-HT3 antagonist isondansetron.
 3. The method of claim 2, wherein the ondansetron isadministered at a dosage ranging from about 1.0 μg/kg per application toabout 5.0 μg/kg per application.
 4. The method of claim 2, whereinondansetron is administered at a dosage of about 3.0 μg/kg perapplication.
 5. The method of claim 2, wherein ondansetron isadministered at a dosage of about 4.0 μg/kg per application.
 6. Themethod of claim 2, wherein ondansetron is administered at least once aday.
 7. The method of claim 2, wherein ondansetron is administered atleast twice a day.
 8. The method of claim 1, wherein the opioid-relateddisorder is selected from the group consisting of: opioid dependence,opioid abuse, opioid intoxication, opioid intoxication delirium,opioid-induced psychotic disorder, with delusions, opioid-inducedpsychotic disorder with hallucinations, opioid-induced anxiety disorder,opioid-related disorder not otherwise specified (NOS), opioidintoxication, and opioid withdrawal.
 9. The method of claim 8 whereinthe 5-HT3 antagonist is ondansetron.
 10. The method of claim 9, whereinthe ondansetron is administered at a dosage ranging from about 1.0 μg/kgper application to about 5.0 μg/kg per application.
 11. The method ofclaim 9, wherein ondansetron is administered at a dosage of about 3.0μg/kg per application.
 12. The method of claim 9, wherein ondansetron isadministered at a dosage of about 4.0 μg/kg per application.
 13. Themethod of claim 9, wherein ondansetron is administered at least once aday.
 14. The method of claim 9, wherein ondansetron is administered atleast twice a day.